Suppression of Human Lens Epithelial Cell Proliferation by Proteasome Inhibition, a Potential Defense against Posterior Capsular Opacification

Abstract

purpose. Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial–mesenchymal transition (EMT) of the remaining lens epithelial cells (LECs) after cataract surgery. Studies have shown that proteasome inhibition interferes with EMT and remodeling of the extracellular matrix. This study was conducted to investigate suppression of LEC proliferation by proteasome inhibition and its signaling pathway.

methods. HLE B-3 cells and human lens epithelium explants from 17- to 20-week fetal lenses were cultured and treated with TGF-β₂ (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL) and 5 or 10 μM MG132. LEC proliferation was determined using both the WST-1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by Western blot analysis. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition.

results. TGF-β₂ suppressed proliferation of HLE B-3 cells, whereas FGF-2 and HGF enhanced proliferation. Proliferation suppression by TGF-β₂ was blocked by adding FGF-2 or HGF. Proteasome inhibitor (MG132) treatment strongly inhibited the proliferation of LECs, either alone or in the presence of TGF-β₂, FGF-2, or HGF. These findings were confirmed by observing PCNA expression. Similar results were obtained with primary human LECs. Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. MG132 caused a significant increase in p21 and p27 protein and decrease in CDK2, but no change in p53, p57, CDK4, or CDK6 protein. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, which reduced p21 and p27 protein expression to very low levels that remained below basal control levels, even after treatment with MG132.

conclusions. Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-β₂, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking development of PCO.

We have reported that proteasome inhibition interferes with two major pathways leading to the development of posterior capsular opacification (PCO): epithelial–mesenchymal transition (EMT) ¹ and remodeling of the extracellular matrix (Wang-Su ST et al. IOVS 2005;46:ARVO E-Abstract 2864). Herein, we report that proteasome inhibition also inhibits LEC proliferation, further supporting the proteasome as a target for prevention of PCO development. 

PCO is the most common postoperative complication of cataract surgery that causes visual loss. ^{2 3} PCO arises from residual LECs at the equator and under the anterior lens capsule after cataract surgery. These cells proliferate and migrate onto the posterior capsule underlying the intraocular lens and into the light path. Many of these cells undergo EMT, resulting in the formation of fibroblasts and spindlelike myofibroblasts, which leads to capsular opacification. ² Therefore, postsurgical medical inhibition of LEC proliferation, migration, and EMT would be a possible option to prevent PCO. In this context, several drugs that can block LEC proliferation, ^{4 5 6} migration, ^{6 7} and EMT ^{8 9} have been studied, but to date none of them has been effective clinically. 

Recent studies have shown the involvement of several cytokines and growth factors such as TGF-β, FGF-2, hepatocyte growth factor (HGF), IL-6, and epidermal growth factor (EGF) in the development of PCO. The levels of these cytokines and growth factors increase in aqueous humor after cataract surgery and influence LEC proliferation, migration and transdifferentiation in the early phase. ^{10 11} Transforming growth factor (TGF)-β has been shown to play a key role in PCO development. TGF-β induces LECs to undergo EMT. ^{12 13 14 15} The resultant transformed cells have fibroblastic and spindle-shaped myofibroblastic morphology, and these cells express proteins that are not normally found in lens cells, such as α-smooth muscle actin, fibronectin, and types I and III collagen. ^{12 16 17} All such changes are associated with human PCO development. In addition, TGF-β has been shown to suppress LEC proliferation. ^{14 18} However, immediately after surgery, when LECs are exposed to increased levels of TGF-β, FGF, and HGF simultaneously, LECs eventually proliferate and undergo changes that result in the formation of PCO. This suggests that FGF and HGF, which have been shown to induce proliferation, may eventually counteract the proliferation suppression of LECs by endogenous TGF-β. 

Acidic FGF (FGF-1) and the more potent isoform, basic FGF (FGF-2), are continuously present in the normal lens environment, and members of the FGF family are known to play a unique role in establishing and maintaining normal lens structure and function. Proliferation, migration, and fiber differentiation of normal LECs have been shown to be affected by FGF-2 in vitro. Previous studies have shown that FGF-2 induces LEC proliferation, ^{19 20} which may contribute to the development of PCO. Also, in explants from weanling rats, FGF-2 counters the effect of TGF-β, ²¹ which otherwise leads to complete loss of cells. ¹³  

HGF is highly expressed in human capsular bag cultures in protein-free medium. ²² Previous studies have shown that HGF induces proliferation of LECs in human LEC lines and in capsular bag cultures. ^{22 23} These findings suggest that HGF plays an important role in the development of PCO. 

Blocking PCO may require inhibition of the remaining LEC proliferation and migration and EMT that occur after surgery. Studies in this laboratory have shown that proteasome inhibition blocks TGF-β-induced EMT markers ¹ and matrix metalloproteinase (MMP) activities (Wang-Su ST et al. IOVS 2005;46:ARVO E-Abstract 2864). The 20S proteasome is a 700-kDa multicatalytic cytoplasmic and nuclear complex that is responsible for most nonlysosomal protein degradation. Proteasomal protein degradation is critical for the regulation of cellular functions, such as cell cycle control, signal transduction, transcription regulation, apoptosis, oncogenesis, migration, antigen presentation, and selective elimination of abnormal proteins. ^{24 25} Targeting the ubiquitin-proteasome pathway represents a novel therapeutic strategy for cancer treatment, with high specificity. ²⁶ The ubiquitin-proteasome pathway regulates TGF-β signaling: degradation of TGF-β receptors and Smads turns off TGF-β signaling, ²⁷ whereas degradation of negative modulators such as Ski and SnoN maintains the signal. ²⁸ In addition, a recent report showed that proteasome inhibitor treatment attenuates FGF-2–induced lens cell proliferation and differentiation. ²⁹  

Recent studies have shown that inhibition of the proteasome exerts an antiproliferative effect by altering the levels of cell cycle regulatory proteins, including p21, p27, p53, p57, CDK2, CDK4, cyclin A, and cyclin B. ^{29 30 31} In the present study we investigated the suppression of LEC proliferation by proteasome inhibition, its signaling pathway, and the effects of added TGF-β₂, FGF-2 and HGF. 

Materials and Methods

Reagents

TGF-β₂ and MG132 were purchased from Sigma-Aldrich (St. Louis, MO); FGF-2 and WST-1 reagent from Roche Diagnostics, Mannheim, Germany; and HGF from PeproTech (Rocky Hills, NJ). The following mouse monoclonal antibodies were purchased: anti-α-tubulin (Sigma-Aldrich), anti-PCNA, anti-p21, and anti-CDK-2 (Santa Cruz Biotechnologies, Santa Cruz, CA). The rabbit polyclonal antibodies (anti-p27, anti-p53, anti-p57, anti-CDK4, and anti-CDK-6) and all the secondary antibodies were purchased from Santa Cruz Biotechnologies. 

Cell Culture and Treatment

The human lens epithelial cell line HLE B-3 was kindly provided by Usha Andley (Washington University, St. Louis, MO). These cells were grown in Eagle’s minimum essential medium, containing 20% fetal bovine serum (FBS), 2 mM glutamine and 50 μg/mL gentamicin at 37°C in a humidified 5% CO₂ atmosphere, unless indicated. The cells were treated with TGF-β₂ (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL), and MG132 (5 or 10 μM), either alone or in combination for 3, 6, or 12 hours. Incubation of LECs for more than 24 hours with MG132 caused increased cell death, as observed by more floating cells. Because of the known limitations of using a cell line, we used low-passage cells and also performed the experiments with cultured primary human LECs. 

Preparation of Lens Epithelial Explants

Under sterile conditions, human fetal lenses of 17 to 20 weeks’ gestational age were dissected from eyes and transferred to Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% FBS, 2 mM glutamine, and 50 μg/mL gentamicin. With fine forceps, the posterior lens capsule was torn and peeled from the fiber cell mass, which was discarded. The remaining lens capsule containing the adherent epithelial monolayer was cut into three pieces, incubated separately in a six-well plate, in DMEM with 20% FBS, 2 mM glutamine, and 50 μg/mL gentamicin, at 37°C in a humidified 5% CO₂ atmosphere. Every piece of the anterior capsule explant showed cell outgrowth within 1 week and resulted in 40% to 60% confluent monolayer cultures within 3 weeks of incubation. All procedures complied with the Declaration of Helsinki and were approved by the Institutional Review Board of the New Jersey Medical School (University of Medicine and Dentistry of New Jersey [UMDNJ]). 

Cell Proliferation Assay

The proliferation of HLE B-3 cells and primary human LECs was evaluated by using a sulfonated tetrazolium salt, WST-1 (4-[3-[4-iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate]). The measurement is based on the ability of viable cells to cleave tetrazolium salts by mitochondrial dehydrogenases. In brief, HLE B-3 cells were plated at a density of 1 × 10⁴ cells/well in 96-well microplates, in phenol red-free medium containing 1% FBS and incubated at 37°C and 5% CO₂. After a 24-hour incubation, at approximately 70% confluence, cells were treated for 12 hours with TGF-β₂, FGF-2, HGF, and MG132, either alone or in combination. To assay for proliferation, 10 μL/well WST-1 reagent was added and incubated for 2 hours at 37°C and 5% CO₂. For primary human LECs, explant cultures were trypsinized and subcultured at a density of 0.5 × 10⁴ cells/well in 96-well microplates in DMEM containing 20% FBS. After 24 hours’ incubation, medium was changed to phenol red-free medium containing 1% FBS and incubated for an additional 24 hours. At approximately 70% confluence, the cells were treated with TGF-β₂, FGF-2, HGF, and MG132, either alone or in combination, and after 24 hours’ incubation, 10 μL/well WST-1 reagent was added, as above, to assay for proliferation. Absorbance of the samples was measured at 450 nm using a microplate reader with a background control as the blank. Induction of proliferation in vehicle-treated control cultures was taken as 1. 

Western Blot Analysis

HLE B-3 cells were grown in 25-cm² flasks and treated, at approximately 70% confluence, with TGF-β₂, FGF-2, HGF, and/or MG132. Human lens epithelial explant cultures were grown in six-well plates and treated when cells covered approximately 50% to 60% of the area in each well. After the desired incubation, cells were washed in PBS and lysed in buffer (25 mM HEPES [pH 7.5], 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.05% Triton X-100, 20 mM β-glycerophosphate, 1 mM orthovanadate, 0.5 mM dithiothreitol [DTT]) supplemented with one protease inhibitor cocktail tablet (Roche Diagnostics) per 10 mL of lysis buffer. The protein concentrations were quantitated by bicinchoninic acid assay (Sigma-Aldrich). The samples containing 10 to 60 μg protein were separated on 12% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked at room temperature for 1 to 2 hours in TBS-T (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat dry milk and incubated overnight at 4°C with anti-PCNA, p21, p27, p57, CDK2, CDK4, or CDK6 antibodies. These blots were then incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies, and specific bands were detected using enhanced chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA) on autoradiographic film. 

RNA Interference Studies

Commercially available small interfering (si)RNA duplexes, directed toward p21 and p27 expression, were purchased from Santa Cruz Biotechnology. Fluorescein-conjugated nontargeted siRNA was used to optimize the transfection conditions and efficiency. Additional controls of nontargeted siRNA duplex or vehicle alone were evaluated and found not to alter p21 and p27 protein expression. Transient transfection of siRNAs was performed using lipophilic transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. Briefly, HLE B-3 cells (1.5 × 10⁵ cells/well) were plated in a six-well plate in medium containing 20% FBS without antibiotics. After 16 hours at approximately 60% confluence, cells were transfected with p21 siRNA (50 nM) and/or p27 siRNA (50 nM), in 8 μL transfection reagent (Lipofectamine 2000; Invitrogen) in a final volume of 1 mL transfection medium (Santa Cruz Biotechnology), and incubated at 37°C for 6 hours. One milliliter growth medium was then added to each well containing transfected cells, without removing the transfection mixture, and incubation was continued for 18 hours. The medium was then replaced with fresh growth medium, and after an additional 24 hours’ incubation, the cells were harvested and total protein was isolated. To evaluate the effect of MG132 in cells transfected with p21 and p27 siRNA, transfected cells were treated with MG132 (10 μM) for the final 6 hours of incubation, before protein extraction. 

Statistical Analysis

Results are expressed as the mean ± SD. Statistical significance was determined by the two-tailed student’s t-test and differences at P < 0.05 were considered as statistically significant. 

Results

Effect of Proteasome Inhibition on LEC Proliferation in Presence of TGF-β₂ and FGF-2

We examined the effect of the proteasome inhibitor MG132 on the proliferation of HLE B-3 cells and primary human LEC cultures, either alone or in the presence of TGF-β₂ and FGF-2, by WST-1 assay and PCNA protein expression. TGF-β₂ (1 ng/mL) suppressed the proliferation of HLE B-3 cells by approximately 21%, and FGF-2 (20 ng/mL) increased proliferation by approximately 35%, within 12 hours of incubation. This small effect of TGF-β₂ and FGF-2 on LEC proliferation observed in our studies is consistent with a previous report. ²⁶ Proliferation suppression by TGF-β₂ was significantly blocked by the addition of FGF-2, so that cell proliferation was comparable to that in control samples. MG132 (5 or 10 μM) treatment strongly inhibited cell proliferation, either alone or in the presence of TGF-β₂ and FGF-2 (Fig. 1A) . These findings were confirmed by the observed expression of PCNA protein, a marker for cell proliferation (Fig. 1B) . Consistent with the WST-1 assay, PCNA expression was decreased by TGF-β₂ and induced by FGF-2. The FGF-2 addition canceled the suppressive effect of TGF-β₂. In addition, MG132 decreased PCNA expression, either alone or in the presence of TGF-β₂ and FGF-2 (Fig. 1B) . Similar but slightly greater changes in cell proliferation were observed with higher doses of TGF-β₂ (10 ng/mL) and FGF-2 (50 ng/mL) (data not shown). In primary human LEC cultures, TGF-β₂, FGF-2, and MG132 treatment caused similar effects on cell proliferation as those seen with HLE B-3 cells (Fig. 1C) . 

Effect of Proteasome Inhibition on LEC Proliferation in the Presence of TGF-β₂ and HGF

Recent studies have shown that HGF also plays an important role in regulating LEC proliferation. We therefore examined the effect of proteasome inhibition on LEC proliferation in the presence of TGF-β₂ and HGF (Fig. 2A) . In our studies, we observed that, similar to FGF-2, HGF (10 ng/mL) induced the proliferation of HLE B-3 cells. Of note, induction in cell proliferation by HGF was greater (80%) than that induced by FGF-2 (35%). HGF addition also cancelled the suppression of proliferation by TGF-β₂. Treatment of HLE B-3 cells with MG132 (5 or 10 μM), either alone or in the presence of TGF-β₂ and HGF, strongly inhibited proliferation (Fig. 2A) . Similar effects on cell proliferation were observed in primary human LEC cultures treated with TGF-β₂, HGF, and MG132 (Fig. 2B) . 

Effect of Proteasome Inhibition on Expression of Cell Cycle Regulatory Proteins

Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. Western blot analysis was performed to observe the expression of p21, p27, p53, p57, CDK2, CDK4, and CDK6 in HLE B-3 cells after treatment with TGF-β₂, FGF-2, and MG132, either alone or in combination, for 3, 6, or 12 hours. We observed that MG132 (10 μM) treatment, either alone or in the presence of TGF-β₂ and FGF-2, caused a dramatic increase in levels of p21 and p27, and decrease in CDK2, but no change in p53, p57, CDK4, or CDK6 proteins, at all incubation times studied. The change in p21, p27, and CDK2 protein levels by proteasome inhibitor treatment was maximum at 6 hours’ incubation. TGF-β₂ and FGF-2 treatment, either alone or in combination, had no significant effect on expression of any of these proteins, suggesting the involvement of some other signaling pathway for their effect on LEC proliferation. The expression of α-tubulin, a housekeeping protein, was used as an internal control (Fig. 3A) . In human fetal lens epithelial explant cultures, as in HLE B-3 cells, MG132 treatment for 6 hours caused an increase in p21 and p27 protein levels, without any significant change in cell morphology (Fig. 3B) . The small change in CDK2 protein levels by MG132 treatment was not investigated further in this study. 

Mediation of the Effect of Increased Levels of p21 and p27 on Antiproliferative Activity of MG132 on HLE B-3 Cells

To verify that increases in levels of p21 and p27, cell cycle inhibitory proteins, by proteasome inhibition mediate the antiproliferation of HLE B-3 cells, siRNA studies were performed. Transfection of HLE B-3 cells with p21 siRNA or p27 siRNA reduced their protein expression by approximately 70% (Fig. 4A) . MG132 treatment (10 μM for 6 hours) of HLE B-3 cells transfected with p21 or p27 siRNA could not increase the levels of these proteins above the basal levels in control cultures (Fig. 4B)demonstrating the effectiveness of the siRNA transfection. Cotransfection of HLE B-3 cells with p21 and p27 siRNA also reduced their protein expression by approximately 70%, and MG32 treatment (10 μM for 6 hours) could not increase the levels of these proteins above the basal level in controls (Fig. 4C) . The expression of α-tubulin, a housekeeping protein, was used as an internal control. 

Next, we determined the effect of MG132 on the proliferation of HLE B-3 cells transfected with p21 and p27 siRNA, either alone or in combination. We found that MG132 treatment strongly inhibited the proliferation of cells transfected either with vehicle or nontargeted siRNA. This antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, either alone or in combination (Fig. 5) . Of note, reducing the expression of p21 and p27 proteins individually, by transfection of cells with p21 or p27 siRNA, did not significantly induce proliferation, compared with controls. This may be due to the involvement of other cell cycle regulatory factors which compensate for the decreased expression of p21 or p27 proteins. 

Discussion

Cataract is the most common cause of vision impairment in the world today. It is treatable with highly effective surgery, but PCO is a common complication of even successful cataract surgery. PCO incidence is approximately 50% in adults and 100% in children. ² Despite several improvements in cataract surgical procedures and intraocular lens (IOL) design, the problem of PCO is still not solved. 

It has been reported that proliferation, migration, and EMT of residual LECs after cataract surgery are the common causes of PCO, and that these processes are influenced by several cytokines and growth factors, including TGF-β, FGF-2, and HGF. Because the proliferation of the remaining LECs may start within a few hours after cataract surgery, inhibition of LEC proliferation may be an effective strategy in preventing PCO. The present investigation was performed to study the suppression of LEC proliferation by proteasome inhibition in the presence of TGF-β₂, FGF-2, and HGF, as a potential strategy to prevent PCO. 

Proteasome inhibitors have been demonstrated to have complex effects on cell cycle regulation, apoptosis, antigen processing and other regulatory pathways, based on the proteasome’s capacity to degrade or process certain short-lived regulatory proteins. The effect of proteasome inhibition depends on cell type, proliferating activity of cells, availability of growth factors, and conditions of treatment. ^{24 25 26} In the present study, we used MG132, a reversible and cell permeable peptide aldehyde inhibitor of the proteasome, as an antiproliferative agent for LECs, to prevent PCO-like changes. An advantage of using MG132 is that it has the ability to inhibit the proteasome reversibly, so that normal cells can recover from its effect, while the challenged LECs that have survived surgery are more likely to be affected. This speculation is supported by the fact that proteasome inhibitor treatment selectively kills tumor cells. ²⁶  

Previous studies have shown more apoptosis in low-density (sparse) cultures than in high-density cultures, which are more resistant to apoptosis. ^{32 33} In our laboratory, we observed that proteasome inhibition had no significant effect on LECs, when treated at high density (confluent monolayer) and was even protective against IFN-γ–induced apoptosis. ³⁴ Based on these facts, we hypothesize that after cataract surgery, when the remaining LEC density is low, proteasome inhibition will have a more antiproliferative effect than it does in high-density confluent monolayer LEC cultures (normal conditions). In addition, proteasome inhibition may have less effect on other ocular tissues that are less damaged after cataract surgery. 

Our findings indicate that MG132 strongly decreases the proliferation of LECs, either alone or in the presence of TGF-β₂, FGF-2, and HGF. Previous reports suggest that TGF-β₂ inhibits proliferation, ^{14 18} whereas FGF-2 and HGF stimulate proliferation, ^{19 20 21 22 23} and therefore after cataract surgery, when the levels of TGF-β₂, FGF-2, and HGF available to LECs increase, ^{10 11} the suppressive effect of TGF-β₂ on LEC proliferation is cancelled by other growth factors such as FGF-2 and HGF. This is believed to lead to normal LEC proliferation and PCO development. Moreover, TGF-β₂ also induces EMT, resulting in fibroblast/myofibroblast cells, further promoting development of PCO. ^{12 13 14 15} Antiproliferative and apoptosis-inducing drugs have been studied with respect to prevention of PCO, but none of them has been effective clinically, probably because of their toxic effects on other ocular tissues and their inability to prevent EMT. Lois et al. ³⁵ recently reported that in a rodent model, TGF-β₂ or anti-TGF-β₂ antibody had no significant effect on PCO development. We speculate that this is due to the differential effects of TGF-β₂ on LEC proliferation and EMT. Thus, a good strategy to prevent PCO is to block proliferation, migration and the EMT of LECs, simultaneously. 

Previous findings in our laboratory indicate that the proteasome inhibitor MG132 blocks TGF-β₂–induced EMT markers in HLE B-3 cells, suggesting that proteasome inhibition can block TGF-β₂-induced EMT. ¹ In addition, residual LEC migration and cell-mediated contraction after wound healing also contribute to PCO. This process is mediated by MMPs. A recent study has shown that MMP inhibition prevents human LEC migration and contraction of the lens capsule, suggesting that MMP inhibition may play a role in preventing PCO. ⁷ Investigations in our laboratory have shown that MG132 decreases MMP-2 and -9 activities in explanted IOLs and HLE B-3 cells, either alone or in the presence of TGF-β₂, suggesting that proteasome inhibition can prevent residual LEC migration and tissue contraction. Therefore, our present finding that a proteasome inhibitor strongly suppresses LEC proliferation in the presence of TGF-β₂, FGF-2, and HGF, together with its ability to block TGF-β₂ induced EMT markers and MMP activities, suggests that the proteasome should be considered as a target for prevention of PCO. 

We observed a strong increase in the protein levels of p21 and p27 by proteasome inhibition, which is consistent with several other reports in the literature. ^{29 30 31} In our studies, when the expression of p21 and p27 proteins was reduced by siRNA transfection, proteasome inhibitor treatment was able to increase p21 and p27 proteins only to levels that remained much lower than those in controls, and the antiproliferative effect of proteasome inhibition was significantly reduced, suggesting that this action requires increased levels of p21 and p27 proteins for maximum effect. 

In summary, the results of this study indicate that proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-β₂, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. Proteasome inhibitors are currently in clinical trial and are approved for treatment of cancer based on their ability to selectively kill cancerous cells. Our study provides additional findings indicating that proteasome inhibitors are good candidates for blocking development of PCO. 

 Supported in part by NEI Grant EY02299 (BJW).

 Submitted for publication February 7, 2006; revised May 22, 2006; accepted August 18, 2006.

 Disclosure: N. Awasthi, None; B.J. Wagner, 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.

 

MG132 inhibited the proliferation of LECs, either alone or in the presence of TGF-β₂ and FGF-2. (A) HLE B-3 cells (1 × 10⁴ cells per well) were cultured in 96-well plates in 1% serum-containing medium. Cells were treated at approximately 70% confluence with TGF-β₂ (1 ng/mL), FGF-2 (20 ng/mL), or MG132 (5 or 10 μM), either alone or in combination. After 12 hours’ incubation, cell proliferation was evaluated with the colorimetric WST-1 assay. (B) HLE B-3 cells grown in 1% serum-containing medium were treated for 12 hours with TGF-β₂ (1 ng/mL), FGF-2 (20 ng/mL), and MG132 (5 or 10 μM), either alone or in combination. Total cell lysates were prepared, proteins were separated by SDS-PAGE, and the gel was immunoblotted with anti-PCNA antibody. (C) Primary human LECs were treated for 24 hours with TGF-β₂ (1 ng/mL), FGF-2 (20 ng/mL), and MG132 (5 or 10 μM), either alone or in combination, and cell proliferation was evaluated with the colorimetric WST-1 assay. (A, C) Data are from one experiment representative of at least three independent experiments with similar results. Data are the mean ± SD of triplicate determinations. *Significant difference compared with control (P < 0.05). (B) Data are from one experiment representative of two independent experiments with similar results.

Figure 2.

 

MG132 inhibits the proliferation of LECs either alone or in the presence of TGF-β₂ and HGF. (A) HLE B-3 cells (1 × 10⁴ cells per well) were cultured in 96-well plates in 1% serum-containing medium. Cells were treated at approximately 70% confluence for 12 hours with TGF-β₂ (1 ng/mL), HGF (10 ng/mL), and MG132 (5 or 10 μM), either alone or in combination. Cell proliferation was evaluated with the colorimetric WST-1 assay. (B) Primary human LECs were treated for 24 hours with TGF-β₂ (1 ng/mL), HGF (10 ng/mL), and MG132 (5 or 10 μM), either alone or in combination, and cell proliferation was evaluated with the colorimetric WST-1 assay. Data are from one experiment, representative of at least three independent experiments with similar results. Data are the mean ± SD of triplicate determinations. *Significant difference compared with the control (P < 0.05).

Figure 3.

 

Western blot analysis of p21, p27, CDK2, CDK4, CDK6, p53, p57, and α-tubulin protein in LECs. (A) HLE B-3 cells were treated for 6 hours with TGF-β₂ (1 ng/mL), FGF-2 (20 ng/mL), and MG132 (10 μM), either alone or in combination. Total cell lysate was prepared, proteins were separated by SDS-PAGE, and the gel was immunoprobed. (B) Human lens epithelial explant cultures were treated for 6 hours with MG132 (10 μM). Total cell lysate was prepared, proteins were separated by SDS-PAGE, and the gel was immunoprobed. The relative expression of proteins was normalized to α-tubulin. Data are from one experiment representative of at least three independent experiments with similar results.

Figure 4.

 

Downregulation of p21 and p27 protein expression by transfection with respective siRNA duplexes. Downregulation of p21 and p27 proteins was specific (A). In cells transfected with p21 and p27 siRNA, either separately (B) or in combination (C), MG132 could only increase p21 and p27 protein levels to values that remained well below basal control levels. HLE B-3 cells were transfected, and after 48 hours, the cells were harvested and protein extracted for Western blot analysis. The relative expression of proteins was normalized to α-tubulin. (A) Cells were transfected with vehicle (lane 1), nontargeted siRNA (lane 2), p21 siRNA (lane 3), or p27 siRNA (lane 4). (B) Cells were transfected with vehicle (lanes 1, 5), nontargeted siRNA (lanes 2, 6), p21 siRNA (lanes 3, 4), and p27 siRNA (lanes 7, 8). The second set of cells transfected with p21 and p27 siRNA were treated with MG132 (10 μM) for 6 hours (lanes 4, 8). (C) Cells were transfected with vehicle (lane 1), nontargeted siRNA (lane 2), cotransfected with p21 and p27 siRNA (lane 3), cotransfected with p21 and p27 siRNA and treated with MG132 (10 μM) for 6 hours (lane 4). (A, C) Data are representative of at least three independent experiments, and each data point represents the mean ± SD (n = 3). (B) Data represent the average of two independent experiments with similar results. p21 siRNA (0.36, 0.25), p21 siRNA+MG132 (0.57, 0.49), p27 siRNA (0.24, 0.3), p27 siRNA+MG132 (0.48, 0.47).

Figure 5.

 

Maximum inhibition of HLE B-3 proliferation by MG132 requires p21 and p27 proteins. HLE B-3 cells grown in 96-well plates were transfected in two sets with vehicle (lanes 1, 2), nontargeted siRNA (lanes 3, 4), p21 siRNA (lanes 5, 6), p27 siRNA (lanes 7, 8) and cotransfected with p21 and p27 siRNA (lanes 9, 10). One set of cells with each transfection was treated with MG132 (10 μM) for 6 hours. After transfection (48 hours), the cell proliferation was evaluated with the colorimetric WST-1 assay. Data are the mean ± SD of triplicate determinations in one experiment, representative of three independent experiments with similar results. (★)Significant difference compared with control (P < 0.05); °significant difference compared with vehicle treated with MG132 (P < 0.05).

The authors thank Harold I. Calvin, PhD, for a critical reading of the manuscript.