Molecular Vision 2005; 11:764-774 <http://www.molvis.org/molvis/v11/a92/> Received 1 March 2005 | Accepted 16 September 2005 | Published 16 September 2005

Download Reprint

Induction of p21^Cip1-mediated G₂/M arrest in H₂O₂-treated lens epithelial cells

Young Seomun,¹ Jong-Tak Kim,¹ Ki-Yong Kim,² Hae-Sook Kim,¹ Ji-Young Park,¹ Choun-Ki Joo¹
 
(The first two authors contributed equally to this publication)
 
 

¹Laboratory of Ophthalmology and Visual Science, Korea Eye Tissue and Gene Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea; ²Central Research Center, Green Cross Corporation, Yongin, Korea

Correspondence to: Choun-Ki Joo, MD, PhD, Laboratory of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-040, Korea; Phone: 82-2-590-2613; FAX: 82-2-533-3801; email: ckjoo@catholic.ac.kr

Abstract

Purpose: Oxidative damage is one of the major factors associated with the formation of age-related cataract and with senescence of various cell types. Although the effects of oxidative stress are complex, we focused on whether oxidative damage affects control of the cell cycle in lens epithelial cells.

Methods: BrdU labeling and FACS analysis were used to investigate the effect of H₂O₂ on the cell cycle of HLE B-3 cells. In addition, western and Northern blot analysis were performed to assess the expression of cell cycle regulatory proteins and transfection with siRNA was used to knock out expression of p21^Cip1. The activation of MAPK family members by oxidative stress was assessed using antibodies to detect the activated forms. To confirm the effect of H₂O₂ on an ex vivo model, its effect on cultures of the lenses of 3-week-old SD rats were examined. The localization and expression of PCNA and p21^Cip1 in the rat lenses were analyzed by immunohistochemistry.

Results: FACS analysis showed that H₂O₂ treatment induced G₂/M phase arrest of HLE B-3 cells. p21^Cip1 was strongly induced by H₂O₂, whereas expression of other cell cycle genes was unchanged. Attenuation of p21^Cip1 expression using siRNA reduced the H₂O₂ induced G₂/M arrest. Furthermore, JNK and ERK were activated by H₂O₂ and their specific inhibitors SP600125 (for JNK) and U0126 (for ERK1/2) prevented p21^Cip1 expression and blocked cell cycle arrest. H₂O₂ treatment of a rat lens organ culture also caused an increase in p21^Cip1. However, H₂O₂ treatment lowered the levels of p27^Kip1, cdc2, and PCNA in the rat lens culture, unlike in the HLE B-3 cells.

Conclusions: The accumulation of p21^Cip1 in lenses exposed to oxidative stress may play a role as a defensive mediator of oxidative damage, an indicator for senescence or aging, or an inducer for the formation of cataract. This finding links oxidative stress with p21^Cip1-mediated control of the cell cycle in lens epithelial cells.

Introduction

Reactive oxygen species (ROS), including the superoxide anion, hydrogen peroxide (H₂O₂), and hydroxyl radicals are natural byproducts of aerobic metabolism [1]. Oxidative stress caused by ROS leads to modification of proteins, lipid oxidation, DNA damage and other impairments of the physiological functions of cells and tissues [2]. It has also been suggested that oxidative stress is an important cause of aging, cancer, senescence (an irreversible cell arrest), and cell death [3].

The lens is derived from ectoderm and grows throughout life. This transparent organ consists of a single layer of epithelial cells on the anterior and equatorial surfaces, and elongated, terminally differentiated cells in the interior. The lens epithelial layer is essential for growth, differentiation, and homeostasis of the entire lens. The lens fiber cells obtain energy and nutrients (including water and ions) through metabolic communication with this monolayer [4]. It has been demonstrated that damage to lens epithelial cells (LECs) as a result of various stresses can be a major cause of the formation of cataract. Oxidative stress, in particular, combined with aging of the lens, is believed to contribute to the formation of age-related cataract [5]. This view is supported by the fact that the level of H₂O₂ is high in the aqueous, vitreous, and lenses of patients with age-related cataract [6]. Moreover, this is correlated with extensive oxidation of the lens components. In addition, H₂O₂ can induce opacification of the lens in rat organ cultures [7,8].

Age-related cataract is a multifactorial disease with a poorly understood etiology. Several lines of evidence suggest that changes in the regulation of the cell cycle in LECs are involved. The proliferative potential of the LECs decreases with age in rodents, suggesting that it is related to telomeric shortening, the induction of senescence, and the appearance of cataracts [9,10]. Furthermore, the abnormal regulation of the cell cycle in the lens, together with the accumulation of damaged proteins, is probably caused by an age-related decline in the ubiquitin-proteasome pathway that is associated with age-related cataract [11]. Despite numerous studies documenting biochemical and metabolic changes in the lens associated with age-related cataract, little is known about the involvement of the cell cycle. Recent reports have attempted to identify changes in gene expression associated with age-related cataract using microarrays [12,13]. Functional clustering of the identified genes revealed that levels of transcripts associated with the control of the cell cycle are reduced in age-related cataract.

Cell cycle arrest plays an important role in development and differentiation [14], and it is a well-known indicator of aging, senescence, and apoptosis [3,15]. Positive regulation of the cell cycle is mediated by the cyclin-dependent kinases (CDKs). Acting in opposition to the CDKs are two families of CDK inhibitors (CKIs); the Cip/Kip family (p21^Cip1, p27^Kip1, and p57^Kip2) and the INK4 family (p15, p16, p18, and p19) [16]. The biochemical activities and patterns of expression of CKIs during development and differentiation implicate these proteins as primary effectors controlling cell cycle exit [17].

Several lines of evidence indicate that temporal and spatial control of the cell cycle is important in differentiation and development of the lens. In the embryonic lens, cell cycle withdrawal is correlated with the expression of p57^Kip2 [18], and with coordinate functioning of p27^Kip1 and p57^Kip2 [19]. Consistent with this view, expression of both p27^Kip1 and p57^Kip2 is upregulated in cultured LECs by inhibition of Src family tyrosine kinases, which causes withdrawal from the cell cycle [20]. In addition, p21^Cip1 and p27^Kip1 are upregulated during bFGF-induced lens cell differentiation [21]. The LECs in the vicinity of the visual axis do not divide, but cell division occurs in the equatorial region and terminal differentiation is initiated in the bow region, which generates the fiber cells. Proper execution of the differentiation program and the formation of mature fibers seem to be essential for lens transparency. This means that abnormalities that result in the incomplete degradation of intracellular organelles are associated with various forms of cataract [12,22].

Because the control of the cell cycle in LECs plays an essential role in lens differentiation, development, and maintenance, an alteration in this control could be directly or indirectly involved in the formation of cataract. Furthermore, the cell cycle can be regulated by oxidative stress, which induces multiphase arrest and subsequent cell death in various types of cells [3,23,24]. These observations, together with the potential effect of ROS as inducers of age-related cataract, prompted us to investigate whether oxidative stress induced by ROS can affect the cell cycle of LECs and, if so, what mechanism is involved. We found that a sublethal dose of oxidative stress to an established line of human LECs (HLE B-3) induced p21^Cip1 expression, followed by G₂/M phase arrest as a consequence of activation of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore, in a rat lens organ culture, lens opacification by H₂O₂ was accompanied by abnormal expression of cell cycle regulators such as p21^Cip1, p27^Kip1, cdc2, and proliferating cell nuclear antigen (PCNA).

Methods

Cell culture conditions and H₂O₂ treatment

Human lens epithelial B-3 (HLE B-3) cells were a kindly provided from Usha P. Andley of Washington University [25]. The cells were cultured in minimum essential medium (MEM, GIBCO, Rockville, MD) with 20% fetal bovine serum (FBS, GIBCO) and 50 μg/ml gentamycin (GIBCO) at 37 °C with 5% CO₂. For experiments, the cells were grown to 70% confluence and then were treated with various concentrations of H₂O₂ (Sigma-Aldrich, St. Louis, MO) in serum free media for the indicated times. For the cell counting experiment, H₂O₂ treated or untreated cells in 35 mm culture dishs were washed with MEM without serum, and then 0.25% Trypsin-1 mM EDTA (Welgene, Dae-gu, Korea) was added for 5 min at room temperature. MEM+20% FBS (1 ml total) was added then the cells were collected. After centrifugation at 2,000 rpm for 5 min, the cells were resuspended in the 1 ml of medium and total cell number was counted. For the inhibitor study, JNK/SAPK specific inhibitor SP600125, MEK inhibitor U0126, p38 inhibitor SB203580, PI-3 kinase inhibitor Wortmanin (Calbiochem. San Diego, CA) and PKC inhibitor GF109203X (Sigma-Aldrich) were added 30 min before H₂O₂ treatment.

5-Bromo-2'-deoxy-uridine (BrdU) Staining

HLE B-3 cells were treated or untreated with 200 μM H₂O₂, incubated for 24 h, and then BrdU was added and incubated for a further 40 min. The cells were fixed with 70% ethanol in 50 mM Glycine pH 2, then immunoflouresence detection of BrdU was performed using the 5-Bromo-2'-deoxy-uridine (BrdU) Labeling and Detection Kit I (Roche Applied Science, Germany) according to the manufacturer's protocol. Nuclear stain was done by addition of Hoechst 33342 (Molecular probes, Eugene, OR).

Flow cytometric cell cycle analysis of HLE B-3 cells

HLE B-3 cells were seeded on 100 mm culture dish at a density of 1x10⁶ cells per dish. The H₂O₂ treated (24 h) or untreated cells were washed twice with ice cold PBS. After treatment of 0.25% Trypsin-1 mM EDTA for 5 min, the cells were collected. The cells were washed twice with PBS then resuspended in 0.5 ml of PBS. Fixation was done by the addition of 5 ml of ice-cold 70% ethanol and incubated overnight at -20 °C. The next day, the cells were washed twice with PBS and resuspended in 500 μl of PBS. After pretreatment with 50 μg/ml of RNase A (Sigma-Aldrich) for 15 min at 37 °C, the cells were stained by addition of 0.5 ml propidium iodide (100 μg/ml in PBS; Sigma-Aldrich) for 30 min at 37 °C. Stained cells were dispersed with a pipette and cell cycle analysis was carried out using a FACSVantage SE (BD Biosciences Immunocytometry Systems, San Jose, CA) using excitation at 536 nm and detection at 617 nm for red fluorescence. The percentage of the cells in each cell cycle phase was determined using the ModFit LT software (Becton-Dickinson) based on the DNA histogram. Ten thousand cells per sample were analyzed.

Antibodies and western blot analysis

Antibodies used in this study were obtained as follows: phospho-JNK, ERK1/2 and JNK from Cell signaling (Beverly, MA). Caspase-3 and p21^Cip1 was from Upstate cell signaling solutions (Charlottesville, VA). p27^Kip1, cdc2, PCNA, caspase-8, caspase-9, PARP, Cyclin A, Cyclin D1, Cyclin E, and phospho-ERK1/2 were from Santa Cruz (Santa Cruz, CA). Actin and α-tubulin were purchased from Sigma-Aldrich. Goat anti-rabbit and anti-mouse antibody conjugated to horseradish peroxidase was purchased from Zymed (South San Francisco, CA).

For western blot analysis, lens epithelial cells were harvested in protein lysis buffer (25 mM Tris-HCl, pH 7.4, 1% Tween-20, 0.1% SDS, 0.5% sodium deoxycholate, 10% Glycerol, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 50 mM NaF, 1 mM Na₃VO₄, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). The cell lysates were incubated on ice for 15 min with occasional mixing and cleared of cell debris and large molecules by centrifugation at 14,000 rpm for 20 min at 4 °C. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL). The lysates containing 10 μg or 20 μg of proteins were boiled for 5 min in 1X SDS sample buffer, loaded and separated on a 10% or 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel, and transferred to a nitrocellulose membrane (Amersham Life Science, Cleveland, OH) using an electrotransfer apparatus (Amersham). Skim milk (5%) in Tris-buffered Saline (50 mM Tris-Cl, 150 mM NaCl, pH 7.4)-Tween-20 (0.1%) was used as a blocking and antibody dilution buffer. The membrane was developed by enhanced chemiluminescence (Santa Cruz). Prestained molecular weight standards were purchased from Elpis-Biotech (DaeJeon, Korea).

Northern blot analysis

HLE B-3 cells were incubated with MEM+1% FBS. The next day, the medium was changed to MEM without serum and then 200 μM of H₂O₂ was added at the indicated times. Total cellular RNA was isolated using TRIZOL reagent (GIBCO). Total RNA was quantified by absorbance at 260 nm. The integrity of RNA was checked on 1% agarose/formaldehyde gel. Total RNA (20 μg) was applied to each lane. The RNA was transferred to a positively charged Nylon membrane (Schleicher&Scuell, Germany) and fixed to the membrane using a UV cross-linker (Stratagene, La Jolla, CA) with exposure of 120 000 μJ/cm². Prehybridization was performed in 5X SSPE, 50% formamide, 10X Denhardt's solution, and 0.5% SDS for 2 h at 42 °C. The membrane was hybridized with p21^Cip1 or β-actin cDNA that was labeled with [α-³²P] dCTP by using a Random Primer DNA Labeling Kit (Roche) for 16 h at 42 °C. The membrane was washed with 2X SSC, 0.1% SDS for 30 min at room temperature, with 0.5X SSC, 0.1% SDS for 30 min at 55 °C, and then twice with 0.2X SSC, 0.1% SDS for 30 min at 55 °C. The membrane was exposed for autoradiography at -70 °C for 4 h on X-ray films.

siRNA experiments

Validated p21^Cip1 specific siRNA was obtained from Ambion (catalog number 51320; Austin, TX). HLE B-3 cells were plated with MEM+1% FBS. At 24 h, the medium was changed to Opti-MEM (GIBCO) and the cells (about 70% confluence) were transfected with the p21^Cip1 siRNAs using SiPORT^TM Lipid siRNA transfection agent (catalog number 4505; Ambion) according to the manufacturer's protocol. H₂O₂ (200 μM) was added after 1 day of transfection. After 24 h of H₂O₂ treatment, the cells were collected and subjected to western or FACS analysis.

Rat lens organ culture and western blot analysis

Whole lenses were carefully removed from 3-week-old male Sprague-Dawley rats and incubated in Medium 199 (Sigma-Aldrich) containing 0.1% BSA (GIBCO) and 50 μg/ml gentamycin at 37 °C with 5% CO₂. The next day, the medium was changed and 200 μM of H₂O₂ was added for 1 day. The entire lens epithelium and fiber cells were carefully removed under the dissection microscope. The isolated lens epithelial cells and fiber cells in 100 μl of lysis buffer wwere extracted by sonication. Protein concentration was determined by BCA assay (Pierce). Total protein (10 or 40 μg) was subjected to western blot analysis.

Rat lens section and immunohistochemistry

For immunohistochemistry, whole rat lenses were fixed in Carnoy's fixative at 4 °C overnight, and then embedded in paraffin. The paraffin-embedded lenses were sectioned on a microtome at a thickness of 7 μm. The lens sections were incubated in 5% normal horse serum and 0.5% BSA in PBS for 1 h at room temperature, then treated with a 1:100 dilution of mouse anti-PCNA or mouse anti-p21^Cip1 (Santa Cruz) for 2 h at room temperature. After 3 washs with PBS, the sections were incubated in 1:200 dilution of anti-mouse Alexa Fluor 488 (Molecular probes) in PBS for 40 min, then counter stained with Hoechst 33258 (Molecular probes). Immunolabeled lens sections were visualized and photographed digitally with an inverted fluorescence microscope (Axiovert S100, Carl Zeiss Meditec) and digital camera (Axiocam, Carl Zeiss Meditec).

Results

H₂O₂ induces cell cycle arrest in HLE B-3 cells

In order to determine the effect of H₂O₂ on cell growth, HLE B-3 cells were exposed to various concentrations of H₂O₂ for 24 h and counted. H₂O₂ (5-200 μM) had only a small effect on cell numbers and no effect on their morphology (data not shown) whereas higher doses (400-800 μM) caused a sharp drop in cell number (Figure 1A). To investigate the effects of sublethal doses of H₂O₂ on the cell cycle, BrdU labeling and FACS analysis were employed, as described in Methods. Cells were treated with 200 μM H₂O₂ for 24 h and fixed following 4 h incubation with BrdU. The cells were then immunostained with anti-BrdU antibody and the ratio of BrdU/Hoechst double-positive cells to the total number of Hoechst-positive cells was determined (Figure 1B). BrdU incorporation was dramatically decreased in the H₂O₂ treated cells, without morphological changes. To determine whether phases of the cell cycle in addition to S phase are affected by H₂O₂ under these conditions, we examined nuclear DNA content by PI staining and FACS analysis (Figure 1C). There was a significant shift toward G₂/M (from 21% to 68.4%), in the H₂O₂ treated cells while the proportion of cells in G₀/G₁ and S decreased (from 48.2% to 13.3% and from 30.8% to 18.3%, respectively Figure 1C, lower). These data demonstrate that treatment of HLE B-3 cells with sublethal doses of H₂O₂ for 24 h induced G₂/M phase arrest.

To test whether H₂O₂ induced cell cycle arrest at a sublethal dose is independent of apoptosis, we examined the activation of caspases and the cleavage of PARP, which is cleaved by activated caspases during apoptosis. As shown as Figure 1D, the expression of caspase-3, caspase-8, and caspase-9 did not change; in addition we did not detect the activated forms of these enzymes, or cleavage of PARP (data not shown). We conclude that 50-200 μM of H₂O₂ induced G₂/M arrest of the HLE B-3 cells without cell death.

p21^Cip1 is induced in H₂O₂ treated HLE B-3 cells

The expression of proteins related to the cell cycle was investigated in H₂O₂ treated HLE B-3 cells by western blot analysis. There were no obvious changes in the expression of cdc2, cyclin A, cyclin D1, cyclin E, p27^Kip1 (Figure 2A), and p57^Kip2 (data not shown) in response to H₂O₂. However, p21^Cip1 increased significantly 8 h after H₂O₂ treatment and remained high for 24 h. Northern blot analysis revealed an increase in p21^Cip1 transcripts 4 h after treatment with 200 μM H₂O₂, reaching a maximum after 16 h (Figure 2B). The data in Figure 2C demonstrate a dose-dependent increase in p21^Cip1 with a maximum at 200 μM, paralleling the G₂/M arrest induced by H₂O₂ (Figure 1C). When cells were exposed to 400 μM H₂O₂, total protein decreased and most of the cells died within 36 h (data not shown).

Regulation of H₂O₂ induced G₂/M phase arrest by p21^Cip1

Accumulating data demonstrate that p21^Cip1 induces G₂/M phase arrest in fibroblast, colorectal cancer cells, and lung cancer cells [23,24]. We therefore asked whether p21^Cip1 is required for H₂O₂ induced G₂/M phase arrest in HLE B-3 cells, using transfection with p21^Cip1-specific siRNA to inhibit the expression of p21^Cip1. Expression of p21^Cip1 in response to H₂O₂ decreased (by 50%) in the siRNA-transfected HLE B-3 cells (Figure 3A). Under these conditions G₂/M phase arrest was partially inhibited (from 69.3% to 48.2%) with a parallel increase in G₀/G₁ phase (from 13.8% to 24.3%; Figure 3B). These results indicated that the H₂O₂ induced expression of p21^Cip1 was responsible for stimulating G₂/M phase arrest in HLE B-3 cells.

JNK and ERK1/2 are involved in the induction of p21^Cip1 by H₂O₂

Many protein kinases and transcription regulatory factors such as MAPK family, PI3K, NF-κB, and AP-1 are activated by oxidative stress [26,27]. To investigate which protein kinases are involved in H₂O₂ induced p21^Cip1 expression and G₂/M phase arrest, we tested specific inhibitors of several signaling kinases known to be activated by H₂O₂; p38 MAPK (SB203580), JNK (SP600125), PI-3 kinase (Wortmanin), MEK (U0126), and PKC (GF109203X). As illustrated in Figure 4A, pretreatment of HLE B-3 cells with 10 μM SP600125 or U0126 blocked the H₂O₂ induced increase of p21^Cip1 protein, suggesting that ERK and JNK, but not the p38 MAPK, PI-3 kinase, nor PKC pathways are involved in this process.

To confirm the involvement of the ERK and JNK pathways in H₂O₂ induced upregulation of p21^Cip1 we examined the kinetics of activation of ERK and JNK. As shown in Figure 4B, we observed bimodal ERK activation in response to H₂O₂. The first peak was rapid and transient, with a maximum at 15 min, followed by a decline toward baseline by 30 min. This was followed by a gradual rise to a secondary sustained rise. In contrast to ERK, JNK activation could be detected within 1 h, reaching a maximum at 2 h and subsequently declining gradually to almost baseline. In addition, p38 MAPK was not activated by H₂O₂ in the HLE B-3 cells (data not shown). Since the timing of the activation of ERK and JNK is closely related with that of p21^Cip1 expression (Figure 2B), these results support the idea that both the ERK and JNK pathways mediate H₂O₂ induced p21^Cip1 expression in HLE B-3 cells.

To see whether the ERK and JNK signaling cascades are actually required for H₂O₂ induced G₂/M arrest, we tested the effect of specific inhibitors of ERK and JNK. As shown in Figure 4C, these inhibitors completely prevented G₂/M phase arrest. We conclude that H₂O₂ induced G₂/M phase arrest in HLE B-3 cells proceeds via p21^Cip1 expression, which is dependent on the ERK and JNK pathways.

The role of p21^Cip1 in H₂O₂ induced cataract formation

To confirm these conclusions ex vivo we used the intact rat lens system, previously described by Spector [8]. When rat lenses were incubated in the presence of 200 μM H₂O₂, opacification was observed in the equatorial region, spreading throughout the superficial cortex (Figure 5A, right) as previously reported. No change in the morphology of the LECs was observed in response to 200 μM (data not shown) but exposure to 400 μM H₂O₂ or more caused severe lens opacity, together with death of LECs (data not shown). In addition, western blot analysis showed that levels of PCNA, cdc2, and p27^Kip1 declined, while p21^Cip1 accumulated dramatically in the rat lens treated with 200 μM H₂O₂ (Figure 5B, left), and the reduction in PCNA and increase in p21^Cip1 were confirmed by immunohistochemistry (Figure 5C). Interestingly, PCNA was present in the differentiated LECs of control rat lenses and disappeared as a result of H₂O₂ treatment. Activation of caspases 3, 8, and 9 was not detected, again suggesting that treatment of rat lenses with 200 μM H₂O₂ does not induce apoptotic cell death (Figure 5B, right).

Discussion

Oxidative stress is believed to be an important cause of aging, cancer, and cell death. It has been also suggested to be a key mediator of the formation of lens cataracts. However, investigating the role of oxidative stress is not simple, as it shuts down some metabolic pathways, increases protein aggregations and oxidation, inhibits ubiquitin-proteasome systems, induces cell senescence/arrest via telomere shortening and/or damage to telomere DNA, stimulates DNA repair systems, activates apoptosis, and mobilizes defenses. Among these complex responses to oxidative stress, the present study focused on control of the cell cycle and regulation of the CKIs in LECs, because it has been suggested that lens maintenance and ocular transparency depend on accurate control of the cell cycle, and that disregulation of the CKIs is associated with cataractogenesis induced by various stimuli. For example, low power microwaves induced G₀/G₁ arrest and p27^Kip1 expression in rabbit LECs [28], and accumulation of p21^Cip1 was observed in a rat sugar cataract model [29]. Recently, Hawse et al. [12] examined human lenses with age-related cataracts and reported a decrease in the levels of cyclin D1 and cyclin G1, which function in the G₀ to S phase transition, and the DNA damage response, respectively. In the present study we found that oxidative stress induced G₂/M phase arrest in the LECs as a result of p21^Cip1 expression, thus providing a link between control of the cell cycle and cataractogenesis.

Oxidative stress may act on growth factor receptors, such as EGFR, to activate the ERK and JNK pathways [27,30,31]. Several lines of evidence indicate that oxidants activate the ERK and/or JNK pathways mainly by stimulating growth-factor receptors, mimicking the actions of natural ligands. This is probably the result of oxidant-mediated inactivation of phosphatases necessary for dephosphorylation of the growth factor receptors [31]. However, we found that pretreatment with AG1478, a specific inhibitor of EGFR, did not inhibit p21^Cip1 expression or ERK/JNK activation by H₂O₂ in HLE B-3 cells (data not shown), indicating that ERK/JNK activation is independent of EGFR transactivation, although we cannot rule out the involvement of other growth factor receptors. Another possibility is that oxidative stress causes the expression and release of cytokine/growth factors such as TGF-β, which activate the MAPK pathways. Because oxidative stress induces the expression of TGF-β [32], we have examined the effect of TGF-β using an antibody that blocks TGF-β function. However, this did not affect p21^Cip1 upregulation and ERK/JNK activation by H₂O₂. Therefore, further studies are needed to investigate how H₂O₂ causes activation of ERK and/or JNK.

p21^Cip1 was originally identified as a gene regulated by the tumor suppressor protein p53 [33], and the induction of p21^Cip1 in response to X-rays and other DNA-damaging agents relies, to different extents, on its transcriptional upregulation by p53 [34]. However, induction of p21^Cip1 in response to mitogenic stimulation and to other stresses occurs via mechanisms that are independent of p53 [34,35]. In this study, we demonstrated that the induction of p21^Cip1 by H₂O₂ in LECs requires both ERK and JNK, but we do not know if it is dependent on p53. The ERK and/or JNK pathways are involved in p21^Cip1 expression in various cell types [36], but there is controversy about how the ERK pathway functions in p21^Cip1 transcription. In p53-deficient human lung cancer cells, H₂O₂ dependent ERK pathways mediate p21^Cip1 upregulation by activating AP-1 and G₂/M phase arrest [24]. JNK, along with ERK, is considered a key mediator of p21^Cip1 expression. JNK-1 responsive cis-acting regulatory elements are present between -127 and -64 of the p21^Cip1 promoter which contains six GC-rich Sp1-responsive elements known to play a major role in p53-independent transcription of p21^Cip1 [37]. Consistent with these results, a previous study showed that c-Jun, a substrate for JNK, mediates the p53-independent activation of the p21^Cip1 promoter by physical interaction with Sp1 [38]. These findings, together with the evidence for ERK/JNK activation by oxidative stress, support the view that H₂O₂ mediated p21^Cip1 induction in the LECs occurs via ERK/JNK activation.

In addition, p21^Cip1 levels are also regulated post-transcriptionally, being subject to proteasome-dependent degradation [39] that can be modulated by interaction with CDKs or PCNA [40,41]. This suggests that the accumulation of p21^Cip1 may be affected by a decrease in proteasome-dependent proteolysis. Normally, LECs and fiber cells possess a fully functional ubiquitin-proteasome pathway and ubiquitin conjugating activity [42,43]. However, it has been reported that a decrease in the ubiquitin conjugating activity in LECs is associated with the accumulation of oxidative damage, aging, and terminal differentiation [11,21,44,45].

The role of p21^Cip1 in oxidative stress remains controversial. Although there is evidence that p21^Cip1 is proapoptotic in certain situations, most studies have provided evidence that it functions as a protective factor during stress, due to its growth-inhibitory properties [46]. Therefore, the accumulation of p21^Cip1 by H₂O₂ may activate a cell cycle checkpoint to rescue the cells from DNA damage. Another role is suggested to be an indicator of senescence in vitro or aging in vivo. At the molecular level, senescence is associated with changes in the expression of a large number of genes [47]. Senescent cells have increased levels of CKIs, p21^Cip1, and p16, which are negative regulators of cell proliferation, and cannot express c-fos, cdc2, and PCNA [48-50]. Furthermore, LECs in age-related cataract also display decreased levels of cyclins [12,13]. Therefore, these data support the idea that accumulation of p21^Cip1 in the LECs is closely related to the formation of age-related cataracts. As in senescence, H₂O₂ treated rat lenses show a decrease in PCNA and cdc2, in contrast to the increase in the p21^Cip1 (Figure 5B). Finally, p21^Cip1 also functions as a transcription cofactor. It can regulate the activity of general transcriptional co-activators (CBP/p300) [51] and repress the activity of well-characterized transcription factors (E2F, c-Myc, and STAT3) [52-54]. Moreover, overexpression of p21^Cip1 is sufficient to induce senescence-like growth arrest in many cell types and the expression of numerous genes associated with senescence and aging [55]. Therefore, it is possible that p21^Cip1 induced by oxidative stress acts as an inducer of age-related cataract.

In the present study, the H₂O₂ treated rat lenses displayed a decrease in the levels of cdc2 and PCNA, and accumulation of p21^Cip1, similar to features of senescence as mentioned above. However, it is not certain that this ex vivo situation is related to the G₂/M phase arrest in rat LECs. Nevertheless, cdc2 plays an important role in entry to mitosis; inhibition of cdc2 activity results in G₂/M phase arrest in various cell types [56] and reduction of its expression. Accumulating data shows that the function of cdc2 is regulated by p21^Cip1, which inhibits cdc2 activity by inactivating CDK2 or PCNA [57,58], and by repressing its transcription together with p53 and Rb [59,60]. Therefore, the accumulation of p21^Cip1 may inhibit cdc2 activity and cause its repression, thus possibly inducing G₂/M phase arrest in the rat lens damaged by oxidative stress. Another point is that unlike the rat lens, H₂O₂ treated HLE B-3 cells did not show any changes in the level of cdc2. HLE B-3 cells were originally immortalized with SV-40 large T antigen [25], which can inactivate p53 and Rb [61]. Therefore, the repression of cdc2 could be attenuated by SV-40 large T antigen due to inactivation of either p53 or Rb in the HLE B-3 cells.

In conclusion, sublethal oxidative stress induces G₂/M phase arrest in HLE B-3 cells via the induction of p21^Cip1, and ERK and JNK pathways regulate the accumulation of p21^Cip1. Furthermore, oxidative damage in intact rat lenses increases accumulation of p21^Cip1, which is accompanied by a decrease in the levels of PCNA, p27^Kip1, and cdc2. A role of p21^Cip1 in the in vitro and ex vivo models is suggested as a defensive mediator against oxidative damage, an indicator of senescence or aging, or an inducer of the formation of cataract. This study provides a pathological mechanism for oxidative stress linked to the control of the cell cycle mediated by p21^Cip1. Further studies will be necessary to elucidate the mechanism underlying ERK and JNK activation by H₂O₂, whether the induction of p21^Cip1 is dependent n p53, and what the exact role of p21^Cip1 accumulation is in the intact lens in response to oxidative damage.

Acknowledgements

We especially thank Jong-Kyu Choi, Sun-Young Park, and Jung-Min Lim for insightful comments and helpful discussions. This work was supported by Korea Research Foundation Grant (KRF-2001-015-FS0017).

References

1. Cerutti PA. Oxy-radicals and cancer. Lancet 1994; 344:862-3.

2. Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 1997; 272:20313-6.

3. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 2000; 408:239-47.

4. Rae JL, Bartling C, Rae J, Mathias RT. Dye transfer between cells of the lens. J Membr Biol 1996; 150:89-103.

5. Spector A. Oxidative stress-induced cataract: mechanism of action. FASEB J 1995; 9:1173-82.

6. Spector A, Garner WH. Hydrogen peroxide and human cataract. Exp Eye Res 1981; 33:673-81.

7. Spector A, Wang GM, Wang RR, Li WC, Kleiman NJ. A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of action. Exp Eye Res 1995; 60:483-93.

8. Spector A, Wang GM, Wang RR, Li WC, Kuszak JR. A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. I. Transparency and epithelial cell layer. Exp Eye Res 1995; 60:471-81.

9. Pendergrass WR, Penn PE, Li J, Wolf NS. Age-related telomere shortening occurs in lens epithelium from old rats and is slowed by caloric restriction. Exp Eye Res 2001; 73:221-8.

10. Wolf NS, Li Y, Pendergrass W, Schmeider C, Turturro A. Normal mouse and rat strains as models for age-related cataract and the effect of caloric restriction on its development. Exp Eye Res 2000; 70:683-92.

11. Shang F, Gong X, Palmer HJ, Nowell TR Jr, Taylor A. Age-related decline in ubiquitin conjugation in response to oxidative stress in the lens. Exp Eye Res 1997; 64:21-30.

12. Hawse JR, Hejtmancik JF, Huang Q, Sheets NL, Hosack DA, Lempicki RA, Horwitz J, Kantorow M. Identification and functional clustering of global gene expression differences between human age-related cataract and clear lenses. Mol Vis 2003; 9:515-37 <http://www.molvis.org/molvis/v9/a65/>.

13. Ruotolo R, Grassi F, Percudani R, Rivetti C, Martorana D, Maraini G, Ottonello S. Gene expression profiling in human age-related nuclear cataract. Mol Vis 2003; 9:538-48 <http://www.molvis.org/molvis/v9/a66/>.

14. Weinberg WC, Denning MF. P21Waf1 control of epithelial cell cycle and cell fate. Crit Rev Oral Biol Med 2002; 13:453-64.

15. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 1993; 90:7915-22.

16. Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol 1999; 39:295-312.

17. Parker SB, Eichele G, Zhang P, Rawls A, Sands AT, Bradley A, Olson EN, Harper JW, Elledge SJ. p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science 1995; 267:1024-7.

18. Lovicu FJ, McAvoy JW. Spatial and temporal expression of p57(KIP2) during murine lens development. Mech Dev 1999; 86:165-9.

19. Zhang P, Wong C, DePinho RA, Harper JW, Elledge SJ. Cooperation between the Cdk inhibitors p27(KIP1) and p57(KIP2) in the control of tissue growth and development. Genes Dev 1998; 12:3162-7.

20. Walker JL, Zhang L, Menko AS. Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases. Dev Dyn 2002; 224:361-72.

21. Guo W, Shang F, Liu Q, Urim L, West-Mays J, Taylor A. Differential regulation of components of the ubiquitin-proteasome pathway during lens cell differentiation. Invest Ophthalmol Vis Sci 2004; 45:1194-201.

22. Pan H, Griep AE. Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development. Genes Dev 1994; 8:1285-99.

23. Barnouin K, Dubuisson ML, Child ES, Fernandez de Mattos S, Glassford J, Medema RH, Mann DJ, Lam EW. H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression. J Biol Chem 2002; 277:13761-70.

24. Chung YW, Jeong DW, Won JY, Choi EJ, Choi YH, Kim IY. H(2)O(2)-induced AP-1 activation and its effect on p21(WAF1/CIP1)-mediated G2/M arrest in a p53-deficient human lung cancer cell. Biochem Biophys Res Commun 2002; 293:1248-53.

25. Andley UP, Rhim JS, Chylack LT Jr, Fleming TP. Propagation and immortalization of human lens epithelial cells in culture. Invest Ophthalmol Vis Sci 1994; 35:3094-102.

26. Finkel T. Redox-dependent signal transduction. FEBS Lett 2000; 476:52-4.

27. Kurata S. Selective activation of p38 MAPK cascade and mitotic arrest caused by low level oxidative stress. J Biol Chem 2000; 275:23413-6.

28. Yao K, Wang KJ, Sun ZH, Tan J, Xu W, Zhu LJ, Lu de Q. Low power microwave radiation inhibits the proliferation of rabbit lens epithelial cells by upregulating P27Kip1 expression. Mol Vis 2004; 10:138-43 <http://www.molvis.org/molvis/v10/a18/>.

29. Takamura Y, Kubo E, Tsuzuki S, Yagi H, Sato M, Akagi Y. Increased expression of p21(WAF-1/CIP-1) in the lens epithelium of rat sugar cataract. Exp Eye Res 2002; 74:245-54.

30. Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ. Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J Biol Chem 1996; 271:4138-42.

31. Chen K, Vita JA, Berk BC, Keaney JF Jr. c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation. J Biol Chem 2001; 276:16045-50.

32. Iglesias-De La Cruz MC, Ruiz-Torres P, Alcami J, Diez-Marques L, Ortega-Velazquez R, Chen S, Rodriguez-Puyol M, Ziyadeh FN, Rodriguez-Puyol D. Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in human mesangial cells. Kidney Int 2001; 59:87-95.

33. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993; 75:817-25.

34. Ewen ME, Oliver CJ, Sluss HK, Miller SJ, Peeper DS. p53-dependent repression of CDK4 translation in TGF-beta-induced G1 cell-cycle arrest. Genes Dev 1995; 9:204-17.

35. Liu Y, Martindale JL, Gorospe M, Holbrook NJ. Regulation of p21WAF1/CIP1 expression through mitogen-activated protein kinase signaling pathway. Cancer Res 1996; 56:31-5.

36. Olson MF, Paterson HF, Marshall CJ. Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1. Nature 1998; 394:295-9.

37. Yu J, Liu XW, Kim HR. Platelet-derived growth factor (PDGF) receptor-alpha-activated c-Jun NH2-terminal kinase-1 is critical for PDGF-induced p21WAF1/CIP1 promoter activity independent of p53. J Biol Chem 2003; 278:49582-8.

38. Kardassis D, Papakosta P, Pardali K, Moustakas A. c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1. J Biol Chem 1999; 274:29572-81.

39. Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, Clurman BE. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol Cell 2000; 5:403-10.

40. Rousseau D, Cannella D, Boulaire J, Fitzgerald P, Fotedar A, Fotedar R. Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway. Oncogene 1999; 18:4313-25.

41. Cayrol C, Ducommun B. Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21. Oncogene 1998; 17:2437-44.

42. Pereira P, Shang F, Hobbs M, Girao H, Taylor A. Lens fibers have a fully functional ubiquitin-proteasome pathway. Exp Eye Res 2003; 76:623-31.

43. Shang F, Gong X, Taylor A. Activity of ubiquitin-dependent pathway in response to oxidative stress. Ubiquitin-activating enzyme is transiently up-regulated. J Biol Chem 1997; 272:23086-93.

44. Hosler MR, Wang-Su ST, Wagner BJ. Targeted disruption of specific steps of the ubiquitin-proteasome pathway by oxidation in lens epithelial cells. Int J Biochem Cell Biol 2003; 35:685-97.

45. Jahngen JH, Lipman RD, Eisenhauer DA, Jahngen EG Jr, Taylor A. Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates. Arch Biochem Biophys 1990; 276:32-7.

46. O'Reilly MA. Redox activation of p21Cip1/WAF1/Sdi1: a multifunctional regulator of cell survival and death. Antioxid Redox Signal 2005; 7:108-18.

47. Cristofalo VJ, Pignolo RJ, Rotenberg MO. Molecular changes with in vitro cellular senescence. Ann N Y Acad Sci 1992; 663:187-94.

48. Stein GH, Drullinger LF, Robetorye RS, Pereira-Smith OM, Smith JR. Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation. Proc Natl Acad Sci U S A 1991; 88:11012-6.

49. Chang CD, Phillips P, Lipson KE, Cristofalo VJ, Baserga R. Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene. J Biol Chem 1991; 266:8663-6.

50. Seshadri T, Campisi J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science 1990; 247:205-9.

51. Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ. Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Science 1997; 275:523-7.

52. Delavaine L, La Thangue NB. Control of E2F activity by p21Waf1/Cip1. Oncogene 1999; 18:5381-92.

53. Kitaura H, Shinshi M, Uchikoshi Y, Ono T, Iguchi-Ariga SM, Ariga H. Reciprocal regulation via protein-protein interaction between c-Myc and p21(cip1/waf1/sdi1) in DNA replication and transcription. J Biol Chem 2000; 275:10477-83. Erratum in: J Biol Chem 2000; 275(21):16400.

54. Coqueret O, Gascan H. Functional interaction of STAT3 transcription factor with the cell cycle inhibitor p21WAF1/CIP1/SDI1. J Biol Chem 2000; 275:18794-800.

55. Chang BD, Watanabe K, Broude EV, Fang J, Poole JC, Kalinichenko TV, Roninson IB. Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proc Natl Acad Sci U S A 2000; 97:4291-6.

56. Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene 2001; 20:1803-15.

57. Smits VA, Klompmaker R, Vallenius T, Rijksen G, Makela TP, Medema RH. p21 inhibits Thr161 phosphorylation of Cdc2 to enforce the G2 DNA damage checkpoint. J Biol Chem 2000; 275:30638-43.

58. Cayrol C, Knibiehler M, Ducommun B. p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene 1998; 16:311-20.

59. Chang BD, Broude EV, Fang J, Kalinichenko TV, Abdryashitov R, Poole JC, Roninson IB. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells. Oncogene 2000; 19:2165-70.

60. Flatt PM, Tang LJ, Scatena CD, Szak ST, Pietenpol JA. p53 regulation of G(2) checkpoint is retinoblastoma protein dependent. Mol Cell Biol 2000; 20:4210-23.

61. Gorman SD, Cristofalo VJ. Reinitiation of cellular DNA synthesis in BrdU-selected nondividing senescent WI-38 cells by simian virus 40 infection. J Cell Physiol 1985; 125:122-6.