Molecular Vision 2002; 8:17-25 <http://www.molvis.org/molvis/v8/a3/> Received 27 November 2001 | Accepted 6 February 2002 | Published 8 February 2002

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Cdk4 and p27^Kip1 play a role in PLC-g1-mediated mitogenic signaling pathway of 18 kDa FGF-2 in corneal endothelial cells

Hyung Taek Lee,¹ Tae Yon Kim,^1,3 EunDuck P. Kay^1,2
 
 

¹Doheny Eye Institute and ²Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA; ³Kon 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 PLC-g1 enzyme activity is essential for cell proliferation in response to FGF-2 stimulation and to investigate the effect of PLC-g1 activation on cell division and on processes that regulate cell cycle progression.

Methods: Cell proliferation was assayed using a colorimetric method to determine the number of viable cells. Subcellular localization of proteins was determined by immunocytochemical analysis, and expression of the proteins was analyzed by immunoblotting. PLC activity was determined by measuring the total inositol phosphates.

Results: When CEC were treated with FGF-2, a prolonged and continuous FGF-2 exposure was required for both PLC enzyme activation and cell proliferation. However, there was a long lag period between the PLC enzyme activation and cell proliferation: the maximum enzyme activity was reached 8 h after FGF-2 stimulation, but no cell proliferation was observed in the cells exposed to FGF-2 for 8 h. Using neutralizing anti-PLC-g1, PLC-b1, or PLC-d1 antibodies, we further demonstrated that PLC-g1 accounts for the hydrolysis of total phosphoinositides (PI) and cell proliferation mediated by FGF-2. The role of PLC-g1 linking to the cell cycle was then determined by the blockades of FGF-2 action on Cdk4 and p27^Kip1. Interestingly, FGF-2 both upregulates Cdk4 synthesis and facilitates the nuclear import of the molecule from the cytoplasm, whereas it facilitates the nuclear export of p27^Kip1 to the cytoplasm without affecting synthesis of the molecule. The neutralizing anti-PLC-g1 antibody completely abolishes the FGF-2 activity on Cdk4, both at the synthetic level and at the level of translocation, and the PLC-g1 antibody blocks the nuclear export of p27^Kip1.

Conclusions: These data indicate that PLC-g1 ultimately leads to activation of the cell cycle machinery to induce cell proliferation mediated by FGF-2. It does so by upregulating Cdk4 expression and by facilitating the nuclear import of the molecule and nuclear export of the Cdk inhibitor (p27^Kip1) to the proteolysis site, the cytoplasm.

Introduction

The corneal endothelium is a monolayer of differentiated cells located in the posterior portion of the cornea. The corneal endothelium is essential for maintaining corneal transparency, but its capacity for regeneration after injury is severely limited in humans and primates [1]. In response to certain pathological conditions, corneal endothelial cells (CEC) in vivo may respond by converting to fibroblast-like cells. These morphologically modulated cells then resume their proliferation ability and start to produce fibrillar collagens, leading to the formation of a fibrillar extracellular matrix. One clinical example of this process is the development of a retrocorneal fibrous membrane [2,3], the presence of which blocks vision, thereby causing blindness. In our previous studies [4,5], corneal endothelium modulation factor (CEMF) secreted by polymorphonuclear leukocytes (PMNs), fibroblast growth factor-2 (FGF-2), or a combination of the two factors was found to modulate phenotypes of CEC, leading to a modulation similar to that observed in vivo. We further found that CEMF could induce de novo synthesis of FGF-2 and that the newly produced FGF-2 is the direct mediator for the modulation of CEC [6].

FGF-2 is a multifunctional regulator of cell development, differentiation, regeneration, senescence, proliferation and migration [7-9]. In normal cornea, FGF-2 is a component of Descemet's membrane that may be necessary for wound repair [6,10-12]. The biological actions of the 18 kDa FGF-2 are mediated through transmembrane cell surface receptors that possess tyrosine kinase activity [13,14]. One of the early cellular events induced by the binding of FGF-2 to its receptor is the stimulation of phosphoinositide (PI)-specific phospholipase C-g1 (PLC-g1) [15,16]. One of the cellular activities mediated by the activated PLC-g1 is mitogenic activity in a variety of cells [16-18]. In our previous studies, we demonstrated that FGF-2 mediates the association of the SH3 domain of PLC-g1 and vinculin [19] and that PLC-g1 associated with cytoskeleton (vinculin and actin) plays a role in mitogenesis [20]. However, to our knowledge, no one has investigated whether the catalytic activity of PLC-g1 is essential for FGF-2-induced mitogenic activity in CEC.

PLC-g1 is involved in the activation of the mitogenic early signaling pathway. This involvement is well understood, but the molecular mechanism that regulates cell proliferation in response to PLC-g1 activation is not. Since cell proliferation is ultimately governed at the level of the cell cycle, it is important to identify the specific components of the cell cycle regulatory apparatus following activation of signaling molecules in response to growth factor stimulation. Strong lines of convergence are emerging in growth signaling. At least several signaling networks appear to be crucial for G₀/G₁ phase progression and G₂/M transition: 1. The mitogen activated protein (MAP) kinase cascade that results in the activation of a range of transcription factors, such as Elk-1, c-Ets-1, and c-Ets-2 [21,22]; 2. The activation of phosphatidylinositol 3 (PI3)-kinase/Akt pathway, which in turn mediates the down regulation of p27^Kip1, thus promoting G₁/S transition of the cell cycle [23-25]; 3. The activation of the MAP kinase cascade that is required for S phase entry and p27^Kip1 downregulation [26]; and 4. The protein kinase C enzymes that appear to operate as regulators of the cell cycle at two sites during G₁ progression and G₂/M transition [27,28].

Transduction of extracellular mitogenic signals culminates in the expression and assembly of different kinase holoenzymes, the cyclin-cyclin-dependent kinase (Cdk) complexes [29,30]. The Cdk complexes are sequentially formed, activated, and inactivated by Cdk inhibitors (CKIs). These sequential steps govern progression through the cell cycle. Two gene families of CKIs have been identified in mammalian cells; the INK4 proteins (inhibitors of Cdk4) and the Kip/Cip inhibitors [30,31]. Overexpression of these inhibitors causes G₁ arrest [31,32]. In the present study, we investigated two crucial aspects of PLC-g1-mediated cell proliferation in response to FGF-2 stimulation: whether PLC-g1 enzyme activity is required for FGF-2-mediated cell proliferation; and whether the activated PLC-g1 has an effect on the processes that regulate cell cycle progression.

Methods

Cell cultures

Rabbit CEC cultures were isolated and established as previously described [4]. Briefly, the Descemet's membrane-corneal endothelium complex was treated with 0.2% collagenase and 0.05% hyaluronidase (Worthington Biochemical, Lakewood, NJ) for 60 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. First passage CEC were used for all experiments. For subculture, confluent cultures were treated with 0.2% trypsin and 5 mM EDTA for 3 to 5 min. Heparin (10 mg/ml) was added to cells treated with FGF-2 (Intergen Co., Purchase, NY), since our previous study showed that CEC require supplemental heparin for FGF-2 activity to occur [5].

Cell proliferation assay

CEC (4x10³) were plated in 96 well tissue culture plates. When cells reached approximately 60% confluency, the media were removed and replaced with serum-free media. After a 24 h incubation, cells were treated with the respective experimental conditions. At the end of the incubation period, 15 ml of CellTiter 96® AQ_ueous One Solution Reagent (Promega, Madison, WI) that contains an MTS tetrazolium compound and an electron coupling reagent (phenazine ethosulfate) was added to each well. The plates were incubated for 2 h at 37 °C in a humidified 5% CO₂ atmosphere. The absorbency was then measured at 490 nm, using the 96 well plate reader.

Measurement of total inositol phosphates

To quantitate total inositol phosphates (IP) formation, CEC (5 x 10⁶) were labeled with myo-(2-³H) inositol (2 mCi/ml, Amersham Pharmacia Biotech, Piscataway, NJ) in inositol- and serum-free DMEM for 24 h at 37 °C in a humidified 5% CO₂ incubator. The labeled cells were washed twice with phosphate-buffered saline (PBS) and incubated at 37 °C for 20 min in serum-free DMEM containing 20 mM LiCl, 20 mM HEPES (pH 7.2) and 1 mg/ml bovine serum albumin (BSA). FGF-2 was then added to the cells and incubation was continued for a designated period of time. The PLC enzyme inhibitor, U73122 (Sigma, St. Louis, MO), was added simultaneously with FGF-2 to study its inhibitory effect. The incubation medium was removed, and the cells were lysed with 0.3 ml of ice-cold 5% HClO₄. After incubation in an ice bath for 30 min, the cell lysates were centrifuged; the supernatant was diluted at a 1:5 ratio with distilled water and applied to an AG1-X8 anion exchange resin (Bio-Rad Laboratories, Hercules, CA). The column was extensively washed with 4 ml of distilled water and then eluted with 10 ml of 60 mM ammonium formic acid containing 5 mM sodium tetraborate. IP was eluted with 2 ml of 1.0 M ammonium formic acid, and 0.1 M formic acid. The radioactivity in the elute was counted using a b-scintillation counter (Beckman Instruments, Inc., Fullerton, CA).

Protein preparation and protein determination

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 [DTT], 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1% Triton X-100) on ice for 30 min. The lysate was subjected to sonication; the cell homogenates were then centrifuged at 15,000x g for 10 min. Concentration of the resultant supernatant was assessed with a Bio-Rad DC protein assay system (Bio-Rad Laboratories).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE): The conditions of electrophoresis were as described by Laemmli, using the discontinuous Tris-Glycine buffer systems [33]. Protein (30 mg) was loaded on a 12.5% SDS-polyacrylamide gel and separated under reduced conditions.

Immunoblotting analysis

The proteins separated by SDS-PAGE were transferred to a nitocellulose membrane (Bio-Rad Laboratories) at 0.22 ampere for 10 h in a semidry transfer system (transfer buffer: 48 mM Tris-HCl, pH 8.3, 39 mM glycine, 0.037% SDS, 20% MeOH). Immunoblot analysis was performed using a commercial ABC Vectastain kit (Vector Laboratories, Inc., Burlingame, CA). All washes and incubations were carried out at room temperature in TTBS (0.9% NaCl, 100 mM Tris-HCl, pH 7.5, 0.1% Tween 20). Briefly, the nitrocellulose membrane was immediately placed in TTBS containing 5% nonfat milk and kept overnight at 4 °C. The incubations were carried out with primary antibodies (1:1000 dilution) for 1 h, with biotinylated secondary antibody (1:5000 dilution; Vector Laboratories Inc.) for 1 h, and with ABC reagent for 30 min. The membrane was treated with the enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech) for 1 min, and the ECL-treated membrane was exposed to ECL film.

Immunofluorescent staining

When cells (5x10⁴) plated in 4-chamber slides reached approximately 80% confluency, they were treated with the respective experimental conditions. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min and simultaneously permeabilized and blocked with buffer containing 0.1% Triton X-100, and 1% BSA in PBS (blocking buffer) for 15 min at room temperature. The subsequent incubation was carried out with blocking buffer and all washes were carried out in PBS at room temperature. Cells were incubated with the primary antibodies (1:500 dilution) for 1 h at 37 °C and then incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:100 dilution) for 1 h at 37 °C in the dark. After extensive washing, the slides were mounted in a drop of Vectashield mounting medium (Vector Laboratories, Inc.) to reduce photobleaching. Control experiments, performed in parallel with the omission of the primary antibodies, showed no activity. For those experiments in which the neutralizing antibodies were added to the culture medium, cells were directly stained with the FITC-conjugated secondary antibody as described above.

Confocal analysis

Antibody labeling was examined using a Zeiss LSM-510 laser scanning confocal microscope. The 1.8 mm optical slices were taken perpendicular to the cell monolayer (apical to basal orientation). A 488 nm Argon laser was used for fluorescein examination, in combination with a 505-530 nm emission filter for detection. A plan-neofluar 25x (N.A. 1.3) oil immersion objective was used to acquire the images. Image analysis was performed using the standard system operating software provided with the Zeiss LSM-510 series microscope.

Antibodies

Anti-PLCg1, PLC-b1, and PLC-d1 antibodies [34] were gifts from Dr. Pann-Guill Suh (Pohang University of Science & Technology, Pohang, Korea). Anti-Cdk4 and p27^Kip1 antibodies were purchased from Sigma and fluorescein-conjugated secondary antibody was purchased from Chemicon (Temecula, CA).

Results

Activation of PLC-g1 by FGF-2

Our previous study demonstrated that FGF-2 mediates the recruitment of PLC-g1 in CEC [20], initially to the membrane and subsequently to the cytoskeleton (actin and vinculin complex). We further showed that the cytoskeleton-associated PLC-g1 is involved in the FGF-2-mediated mitogenic signaling pathway: cytochalasin B inhibited the association of PLC-g1 to the cytoskeleton and subsequently inhibited cell proliferation in response to FGF-2 stimulation [20]. However, we did not investigate whether the enzymatic activity of PLC-g1 is required for FGF-2-induced DNA synthesis in CEC. To determine whether FGF-2 induces hydrolysis of phosphatidylinositol phosphates in CEC, cells were starved in inositol- and serum-free medium and labeled with [³H] myoinositol. The cells were incubated in the presence of FGF-2 (10 ng/ml) for a designated period of time, and radioactivity in total IP was measured by anion exchange chromatography. Cells were initially exposed to the growth factor for 5 min to observe the early effect; exposure time was then increased by 5 min increments to a total of 30 min. As shown in the cells stimulated for 30 min, there is no increase in total PI hydrolysis (Figure 1A). Therefore, exposure time was prolonged up to 48 h. Cells treated for at least 1 h showed a twofold increase in enzymatic activity; PLC activity peaked after 8 h of FGF-2 stimulation (almost a 13 fold increase from the basal level); thereafter, enzyme activity decreased. Cells treated with FGF-2 for 48 h showed a marked reduction in enzyme activity. This observation was confirmed by the in vitro PLC enzyme assay result (data not shown). It should be noted that the basal PLC activity in CEC is negligible in the absence of FGF-2 stimulation. Since a 1 h incubation of cells with FGF-2 is sufficient to stimulate PLC enzyme activity, we determined the dose-response effect of FGF-2 on total PI hydrolysis after one-hour incubation with the growth factor. Figure 1B shows that FGF-2 at 1 ng/ml can generate an approximate twofold increase in enzyme activity and that higher doses of FGF-2 did not promote a significant increase in enzyme activity.

To determine whether PLC enzyme activity correlated with cell proliferation, cell proliferation was assayed after a 24 h treatment with FGF-2 in concentrations ranging from 0.01 ng/ml to 50 ng/ml. At a concentration of 1 ng/ml, FGF-2 showed a nearly maximal stimulatory activity on cell proliferation. The higher concentrations of the growth factor did not significantly stimulate further cell proliferation (Figure 2A). Cell proliferative activity was also measured as a function of exposure time to the growth factor (Figure 2B). Cells stimulated with FGF-2 for 30 min, for two duplicated 30 min exposures with an 8 h interval, or for a continuous 8 h exposure showed no proliferative activity. Cells treated with FGF-2 for 24 h demonstrated a marked increase in cell proliferation, suggesting that cell proliferation requires a prolonged and continuous FGF-2 exposure in CEC. This finding confirms our previous findings that a 16 h exposure did not results in proliferative activity and that CEC require a 24 h exposure to FGF-2 to stimulate cell proliferation [20]. These findings are consistent with those of other researchers [35,36], which showed that platelet-derived growth factor (PDGF)- or epidermal growth factor (EGF)-stimulated cell cycle progression requires the continuous presence of these growth factors. The long lag period between the activation of PLC enzyme activity and cell proliferation in CEC is interesting; although PLC enzyme activity reached maximal levels after 8 h of FGF-2 exposure (Figure 1A), no cell proliferation activity was noted in cells treated with FGF-2 for 8 h (Figure 2B).

To confirm that total PLC enzymes are involved in endothelial cell proliferation mediated by FGF-2, the effect of U73122, a specific inhibitor of PI-PLC, was determined in concentrations ranging from 1.0 to 40 mM (Figure 3A). U73122 inhibited cell proliferation mediated by FGF-2 in a dose-dependent manner; no significant inhibitory actions were observed up to 10 mM, but there was a marked inhibition of cell proliferation at 20 mM and 40 mM of the inhibitor. The effect of different concentrations of U73122 ranging from 1.0 mM to 40 mM on PLC enzyme activity was also determined (Figure 3B). U73122 was found to have a dose-dependent inhibitory effect on FGF-2 stimulated PLC activity; at concentrations up to 10 mM, there was a gradual increase of inhibitory action; higher concentrations produced approximately 50% inhibition.

Since U73122 blocks the whole spectrum of PI-PLC enzymes, including PLC-band PLC-d, a specific antibody made against PLC-g1 was used to block PLC-g1 activity (Figure 4A). In the absence of FGF-2 and anti-PLC-g1 antibody, the basal PLC activity in CEC was very low, consistent with the results shown in Figure 1A and Figure 3B. Cells treated with FGF-2 alone showed an approximate 7.5-fold increase in PLC-g1 activity. When cells were treated simultaneously with FGF-2 and anti-PLC-g1 antibody, 0.5 ml of the neutralizing antibody was able to block the enzyme activity by 20% and 5 ml of the undiluted antibody inhibited approximately 25% of PLC-g1 activity (Figure 4A). The effect of anti-PLC-g1 antibody on cell proliferation mediated by FGF-2 was also determined; cell proliferation was assayed after cells were treated for 24 h with FGF-2 at 10 ng/ml, with simultaneous treatment with anti-PLC-g1 antibody. The neutralizing antibody inhibited cell proliferation in a dose-dependent manner: no significant inhibitory action was observed in the cells treated with 0.5 ml of the undiluted antibody, but 5 ml of the neutralizing antibody inhibited cell proliferation by 20% (Figure 4B). This level of inhibition of cell proliferation, which is directly mediated by PLC-g1 action, is in agreement with our previous study, as determined by PLC-g1 specific antisense oligonucleotide primer [20]. Since PLC-b and PLC-d are also known to contribute to the growth factor-stimulated mitogenic signaling pathway [34,37], neutralizing antibodies for PLC-b1 and PLC-d1 were tested to determine whether they inhibited PI hydrolysis in response to FGF-2 stimulation. Whereas anti-PLC-g1 antibody inhibits PI hydrolysis, neither anti-PLC-b1 antibody nor anti-PLC-d1 antibody had this same effect (Figure 4C). These data suggest that PLC-g1 mediates PI hydrolysis in response to FGF-2 stimulation in CEC and that FGF-2 does not use the PLC-b- or PLC-d-mediated signaling pathway for its mitogenic activity.

To confirm whether the antibodies that were exogenously added to the culture medium were taken up by the cells, the endocytosed anti-PLC antibodies were stained with FITC-conjugated secondary antibody. Cells maintained in DMEM-10 in the absence of the exogenously added neutralizing antibody showed negative staining (Figure 5A). Cells that took up the antibodies demonstrated strong cytoplasmic staining patterns for anti-PLC-g1 antibody (Figure 5C) and anti-PLC-d1 antibody (Figure 5D). The internalized anti-PLC-b1 antibody, on the other hand, is localized in the nuclei (Figure 5B). These data indicate that CEC are phagocytically competent, similar to fibroblasts that actively take up collagen molecules [38]. The staining pattern for the endocytosed PLC-g1 antibody and PLC-d1 antibody further showed that the internalized antibodies are not targeted to lysosome. We have reported that lysosomes in CEC show the characteristic punctate globular profile when stained with anti-lysosomal associated membrane protein 2 antibody [39]. These data suggest that the endocytosed antibodies are not intracellularly degraded for at least 24 h after uptake. Furthermore, these findings are in agreement with our previous report, in which the exogenously added neutralizing antibodies have conducted their blocking action inside the cells [19].

Linking PLC-g1 to the cell cycle

Although the involvement of PLC-g1 in the activation of the mitogenic signaling pathway mediated by growth factors is well known, the molecular mechanism regulating cell proliferation in response to PLC-g1 activation is not understood. Progression of the cell cycle in all eukaryotic cells depends on the activity of a series of kinase complexes composed of cyclins and Cdks. The activity of cyclin-Cdk complexes is regulated by various mechanisms, including association of the complex with a group of Cdk inhibitors. Therefore, we examined the expression of cell cycle regulatory proteins in response to FGF-2 stimulation, using indirect immunofluorescent staining. Cyclin D1, cyclin E, Cdk2, and p21^Cip1 were not detectable, either in cytoplasm or in CEC nuclei (data not shown). Unlike these proteins, which demonstrate a negative staining potential, Cdk4 demonstrated a diffuse cytoplasmic and perinuclear staining in cells that were maintained in DMEM-10 for 3 days and then deprived of serum for 24 h prior to staining (Figure 6B). Interestingly, FGF-2 induced translocation of Cdk4 into the nuclei (Figure 6C), and anti-PLC-g1 antibody completely abolished the nuclear import of the protein (Figure 6D), suggesting that PLC-g1 may be involved in the nuclear import of Cdk4. Cells stained in the absence of primary antibody showed a negative staining (Figure 6A). When expression of p27^Kip1 was examined, cells deprived of serum for 24 h showed a strong nuclear staining of p27^Kip1 (Figure 7B), but cells stained in the absence of anti-p27^Kip1 antibody had a negative staining profile (Figure 7A). Interestingly, FGF-2 induces nuclear export of p27^Kip1 to the cytoplasm, resulting in a dual subcellular localization of the protein at nuclei and cytoplasm (Figure 7C). Such translocation of p27^Kip1 is probably prerequisite for ubiquitin-mediated proteolysis. The neutralizing anti-PLC-g1 antibody completely abolished the nuclear export of p27^Kip1 (Figure 7D), suggesting that PLC-g1 may play a role in the degradation pathway of p27^Kip1 by facilitating nuclear export.

When expression of Cdk4 and p27^Kip1 was further examined using immunoblotting analysis, cells deprived of serum for 24 h showed a basal level of 34 kDa Cdk4, whereas the Cdk4 level was markedly stimulated by FGF-2 (Figure 8A). Such stimulation of Cdk4 expression was inhibited by anti-PLC-g1 antibody, suggesting that PLC-g1 may also be involved in the synthesis of Cdk4. Unlike the Cdk4 expression, the expression level of p27^Kip1 is not affected by either FGF-2 or the neutralizing PLC-g1 antibody (Figure 8B).

Discussion

We have previously shown that FGF-2 mediates the recruitment of PLC-g1, initially to the membrane and subsequently to the cytoskeleton [20]. The PLC-g1 associated with cytoskeleton is involved in the FGF-2-mediated mitogenic signaling pathway. However, the question of whether the catalytic activity of PLC-g1 is essential for FGF-2-induced mitogenic activity in CEC has not been studied. There have been conflicting reports about whether PLC-g1 enzyme activity is required for cell proliferation in response to growth factor stimulation: Wang et al. found that PLC-g1 enzyme activity is essential for both EGF- and PDGF-induced DNA synthesis in MDCK cells and NIH 3T3 cells [40], whereas Smith et al. reported that PLC-g1 can induce DNA synthesis by a mechanism independent of its lipase activity [17]. Therefore, we examined whether the enzymatic activity of PLC-g1 is required for FGF-2-induced mitogenesis.

In the present report, we demonstrated that PLC-g1 enzyme activity is required for CEC proliferation in response to FGF-2 stimulation: FGF-2 significantly stimulates PLC enzyme activity in a dose-dependent manner and stimulates cell proliferation in a dose- and time-dependent manner. U73122 is a specific PI-PLC inhibitor that markedly blocks total PLC enzyme activity and cell proliferation. Furthermore, the specific anti-PLC-g1 antibody inhibits total PI hydrolysis by 25% and cell proliferation approximately by 20% in response to FGF-2 stimulation. Unlike the neutralizing PLC-g1 antibody, neither anti-PLC-b1 antibody nor anti-PLC-d1 antibody inhibits hydrolysis of total PI, suggesting that PLC-g1 is responsible for the hydrolysis of total PI to generate the second messengers that ultimately induce cell proliferation in response to FGF-2 stimulation. One unique and important finding is the long lag period between the initial burst of PLC enzyme activity and cell proliferation: the activation of PLC enzyme activity requires at least a 1 h stimulation with FGF-2, and maximum PLC enzyme activity is reached 8 h after FGF-2 stimulation. However, the subsequent cell proliferation requires prolonged, continuous stimulation with FGF-2 (at least 24 h). This finding is in agreement with the previous report, in which prolonged, continuous PDGF exposure results in S-phase entry many hours after the initial burst of PI3-kinase activity [35]. This previous study provides further evidence that PDGF induces both an early and a late wave of PI3-kinase activity and that only the late wave is required for progression through G₁. To the contrary, the present study shows that FGF-2 does not induce two waves of PLC-g1 activity in CEC, suggesting that there may be differential stimulation of signaling enzymes in response to the respective growth factors.

Another unique finding in the present study is that the maximum inhibition of PLC-g1-mediated cell proliferation in response to FGF-2 stimulation is only 15% to 25% of the level of FGF-2-mediated cell proliferation: neutralizing anti-PLC-g1 antibody is able to block approximately 20% of cell proliferation (Figure 4B). Our previous study reported that both PLC-g1 specific antisense oligonucleotide primer and cytochalasin B, which blocks the association of PLC-g1 with the cytoskeleton, inhibit the mitogenic activity of FGF-2 by 15% to 25% [20]. Together, these findings suggest that the mitogenic signaling pathway via PLC-g1 may be one of the several pathways used by FGF-2. We have shown that PI3-kinase is also involved in the mitogenic signaling pathway in response to FGF-2 stimulation in CEC [20].

Although FGF-2 stimulation requires the activation of cytoplasmic signaling molecules, such as PLC-g1, to induce cell proliferation, the resultant signaling must ultimately lead to activation of the cell cycle machinery for cells to progress through the G₁ phase of the cell cycle and into the S phase. The present study further explores whether PLC-g1 takes part in the specific stage in the cell cycle, in particular attempting to identify the specific components of the cell cycle regulatory apparatus after PLC-g1 activation. In the present study, we demonstrate that PLC-g1 plays a part in the upregulation of Cdk4 expression at two respective regulatory steps: first, FGF-2 stimulates the synthesis of Cdk4, and anti-PLC-g1 antibody blocks the stimulatory activity of FGF-2 on Cdk4 expression; second, and most interestingly, FGF-2 induces the nuclear import of Cdk4, and anti-PLC-g1 antibody completely abolishes the nuclear import of the molecule. Considering the crucial role of the nuclear localization of Cdk4 for the active complex formation with cyclins during the G₁ phase and G₁ to S phase transition, translocation of the molecule from the cytoplasm (the biosynthetic site) to the nuclei (the action site) may be an early regulatory step of Cdk4 activation during cell cycle progression. Our data suggest that this crucial step is mediated by PLC-g1 in the mitogenic signaling pathway of FGF-2. A further study to investigate whether PLC-g1 induces the association of Cdk4 with a cyclin subunit (mostly D type) and phosphorylation of Cdk4 is under way. Furthermore, the molecular mechanism by which PLC-g1 exerts such action needs to be elucidated.

Another interesting finding of the present study is that FGF-2 induces the nuclear export of p27^Kip1 into cytoplasm, and anti-PLC-g1 antibody completely blocks FGF-2 action on the translocation of p27^Kip1. The nuclear export of p27^Kip1 is prerequisite for the ubiquitination of the molecule and the subsequent degradation at the proteasome. It has been reported that p27^Kip1 is largely regulated at the posttranslational level (proteolysis). The mitogen-regulated decrease of p27^Kip1 expression occurs through a degradation mechanism in the proteasome [41]. It has also been reported that PDGF decreases the level of p27^Kip1 and that MAP kinase is required for p27^Kip1 downregulation and S phase entry in fibroblasts and epithelial cells [42,43]. In NIH 3T3 cells, the addition of wortmannin blocks EGF-dependent loss of p27^Kip1 and S-phase entry, suggesting that PI3-kinase contributes to G₁ progression, at least in part, by stimulating the degradation of the Cdk inhibitor [44]. Our data indicate that neither FGF-2 nor PLC-g1 is directly involved in the synthesis of p27^Kip1 in CEC that reach confluence. Instead, PLC-g1 facilitates the nuclear export of p27^Kip1 for the subsequent ubiquitin-mediated proteolysis. Taken together, our data indicate that PLC-g1 acts as a direct mediator, linking the cell cycle to mitogenic activity of FGF-2 while facilitating the translocation of Cdk4 and p27^Kip1 between the cytoplasm and the nuclei in corneal endothelial cells.

Acknowledgements

Support for this work was provided by NIH grants EY06431 and EY03040.

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