Alteration of Cadherin in Dexamethasone-Induced Cataract Organ-Cultured Rat Lens

Abstract

purpose. A side effect associated with long-term treatment of various diseases with steroids is a high incidence of posterior subcapsular cataracts (PSC). To understand the mechanism underlying steroid-induced cataract, the cultured lens model was developed, and the expression of potential candidate proteins during opacity formation was examined.

method. Rat lenses were carefully dissected from the surrounding ocular tissue and incubated in medium 199. Dexamethasone was then added to the medium. The lenses were cultured for 7 days and photographed daily to record the development of opacity. Differential expression of candidate proteins was examined by Western blot analysis.

result. Various degrees of opacity were observed on the posterior subcapsular region as early as 5 days after incubation with dexamethasone. The expression of E-cadherin and N-cadherin decreased in the cultured rat lenses during the development of opacity.

conclusions. The pattern of opacity that developed in cultured rat lenses closely resembled that observed in patients with PSC. The results suggest that the decrease in E-cadherin plays a role in the formation of steroid-induced cataract.

Long-term systemic treatment with steroids results in a high incidence of posterior subcapsular cataract (PSC). ^{1 2} However, certain patients cannot avoid long-term steroid therapy. Despite various studies of steroid-induced cataracts, the precise molecular mechanism involved in the formation of steroid-induced cataract is unclear, because it is difficult to procure human samples. To understand the mechanism underlying steroid-induced cataracts, an animal model system replicating the steroid effect on the human lens was developed. A few animal models have been developed to examine the effects of glucocorticoids. After an intravitreous injection of glucocorticoid, opacification appeared in rabbit lenses below the posterior capsule. ³ Long-term administration of steroids to rats induced cataracts in rats similar to human steroid-induced cataracts. ⁴ However, it takes more than 8 months to produce phenotypic changes with the in vivo model system. One of the mechanisms suggested for PSC formation is that inhibition of Na⁺K⁺-adenosine triphosphatase (ATPase) by corticosteroid increases intracellular sodium concentrations and decreases potassium levels, which leads to the accumulation of water within lens fiber cells. ⁵ Several reports have shown that the formation of Schiff bases between the steroid C-20/C-21 carbonyl groups and the ε-amino groups of lysine residues in crystallin is involved in the destabilization of protein structures and allows further protein modification, which leads to cataracts. ^{6 7 8} Nishigori et al. ⁹ showed that the level of glutathione decreases in the lens during steroid therapy, and that the level of lipid peroxide increased in the lens ¹⁰ and liver. ¹¹ These results suggest that steroid-induced cataract formation is caused by oxidative stress, rather than by the formation of a Schiff base. ¹²  

Cadherin, a family of cell-cell adhesion molecules, controls calcium-dependent cell adhesion. Cadherin is necessary for formation of cell-cell junctions, which involves adherence junctions and tight junctions. ¹³ Because the absence of cell adhesion molecules induces cataracts, ¹⁴ cell-cell adhesion may play an important role in maintaining lens transparency. Previous studies have demonstrated that dexamethasone inhibits the expression of N-cadherin in human osteoblastic cells and E-cadherin in human bronchial epithelial cells. ^{15 16} Furthermore, t-butyl hydroperoxide-induced oxidative stress disrupts the E-cadherin-catenin cell-adhesion complexes in mouse liver slices. ¹⁷ However, whether cadherin expression is affected during formation of steroid-induced cataract has not been investigated. 

In this report, steroid-induced cataracts were simulated in an ex vivo model, in which rat lenses were incubated with dexamethasone. Opacity was induced in the posterior region of the rat lenses and was morphologically similar to human steroid-induced cataracts. Moreover, E- and N-cadherin protein levels decreased during the development of opacity. The glucocorticoid receptor (GR) antagonist RU486 inhibited the development of opacity induced by dexamethasone. These results suggest that the GR-mediated reduction of cadherin can have a potential role in treatment of for steroid-induced cataracts. 

Materials and Methods

Ex Vivo Rat Lens Culture and Drug Treatment

All animal procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For rat lens culture, eyes from 21-day-old Sprague-Dawley male rats were enucleated and placed in serum-free M199 medium (pH 7.2; Sigma, St. Louis, MO), containing bovine serum albumin (BSA; GibcoBRL, Grand Island, NY) and antibiotic solution (GibcoBRL; 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B). Rat lenses were cultured as reported previously. ¹⁸ The eyes were opened posteriorly to avoid damaging the lens, and pressure was applied to the anterior segment to expel the lens using plastic-coated forceps and fine scissors. Lenses were immediately transferred into culture medium, which was changed daily. Approximately 24 hours after the preparation of organ cultures, clear lenses were selected, and dexamethasone (Sigma) was added to a final concentration of between 1 and 5 μM. RU486 (Sigma), a GR antagonist, or both dexamethasone and RU486 were added to the culture medium at final concentrations of 5 μM to block glucocorticoid signaling. Fluorescently labeled 5 μM dexamethasone (dexamethasone fluorescein; Molecular Probes, Eugene, OR) dissolved in dimethyl sulfoxide (DMSO; Sigma) or DMSO alone was added to the cultures. Lenses were cultured for 7 days at 37°C under 5% CO₂. Lenses were observed daily under a stereomicroscope and photographed. 

Histologic Analysis

At the end of the culture period, the lenses were placed in phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde. The lenses were dehydrated, embedded in paraffin, and cut into 5-μm sections. For histology, dewaxed paraffin sections were washed with Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl [pH 7.4]) and incubated for 40 minutes in TBS containing 0.05% Triton X-100, 2% BSA, 0.1 μg/mL ethidium bromide (Molecular Probes), and 1 μg/mL 3,3′-dihexyloxacarbocyanine iodide (DiOC₆; Molecular Probes). The sections were then washed with TBS for 30 minutes and mounted in aqueous medium (Aqua Poly/Mount; Polysciences, Eppelheim, Germany). To visualize the nuclei and membranes, ethidium bromide and DiOC₆ were used, respectively. 

For immunohistochemistry, flat preparations of the equatorial capsule (containing adherent epithelial cells) were prepared by bisecting the lens and removing lens fibers. These were fixed for 20 minutes at room temperature in 4% paraformaldehyde in PBS. They were then blocked in PBS containing 5% horse serum and 0.1% Triton X-100, and incubated overnight with polyclonal anti-GR (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500. Samples were then incubated for 1 hour with rhodamine-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:500. Histologic images were photographed with a microscope (Axiovert model S100; Carl Zeiss, Oberkochen, Germany) and a digital camera (AxioCam; Carl Zeiss). 

For electron microscopy (EM) studies, the lenses were fixed in 4% glutaraldehyde (0.1 M sodium phosphate buffer, pH 7.4) and postfixed in 1% osmium tetroxide. The tissues were washed and dehydrated through a graded series of ethanol, cleared in propylene oxide, embedded in Epon mixture, and sectioned at 1 μm and 90 nm with an ultramicrotome. Semithin sections (1 μm) were stained with toluidine blue for light microscopy. Thin sections (90 nm) were stained with uranyl acetate and lead citrate, and observed by transmission electron microscopy (TEM; model H-600; Hitachi, Ltd., Tokyo, Japan). 

Determination of GSH in the Lens

Dexamethasone-treated lenses and control lenses were homogenized in cold phosphate-EDTA buffer, and centrifuged to collect the supernatant. Glutathione (GSH) levels were estimated by a process based on the method of Lou et al. ¹⁹ The lens extracts were added to trichloroacetic acid (TCA) and centrifuged, and supernatants were used for the determination of GSH. 

RT-PCR Analysis

Total RNA was prepared from whole lenses with extraction reagent (Trizol; GibcoBRL). Total RNA (2 μg) was used for reverse transcription with reverse transcriptase (SuperScript II; Gibco BRL). PCR amplification was performed with reverse-transcribed DNA, 10 μM specific primers, 0.2 mM dNTP, and 1 mM MgCl₂. The reaction was performed for 30 cycles of denaturation at 94°C for 50 seconds, annealing at 57°C for 50 seconds, and extension at 72°C for 1 minute. PCR primer sequences and the sizes of the amplified products for each cDNA were as follows: E-cadherin, 5′-GACATCATCACTGTGGCAGC-3′ and 5′-TCTGCAGCAACAGTGTGGAC-3′, 410 bp; N-cadherin, 5′-ACACTCAAGGTGACTGA-3′ and 5′-CGCGCAGTGTAAGATGCGAT-3′, 350 bp; β-catenin, 5′-TTCAGCTGCTTGTACGAGCA-3′ and 5′-CATTCCTGGAGTGGAGCAAC-3′, 336 bp; and β-actin, 5′-AGGCCAACCGCGAAGATGACC-3′ and 5′-GAAGTCCAGGGCGACGTAGCAC-3′, 350 bp. 

Western Blot Analysis and Preparation of Subcellular Fractions

Lenses were rinsed with PBS on ice and then homogenized directly in 1.5-mL conical plastic tubes on ice in an appropriate volume of lysis buffer (50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 10% glycerol, 100 mM NaCl, 1 mM EDTA, 20 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin). Homogenates were clarified by centrifugation at 14,000g for 15 minutes at 4°C. The protein concentration of each sample was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Protein (30 μg) from each sample was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose membranes. Blots were incubated for 1 hour at room temperature in TBS containing 0.1% Tween 20 (TBST) and 5% nonfat dried milk. They were then incubated overnight at 4°C with primary antibodies directed against the corresponding target proteins, diluted in TBST and 5% nonfat dried milk, according to the manufacturer’s (Pierce) recommendations. Mouse monoclonal anti-E-cadherin (Transduction Laboratories, Oxford, UK), mouse monoclonal anti-N-cadherin (Transduction Laboratories), mouse monoclonal anti-β-catenin (Transduction Laboratories), and mouse monoclonal anti-actin (Sigma) antibodies were used to detect the corresponding proteins. Membranes were washed in TBST (35 minutes), incubated for 40 minutes in TBST and 5% nonfat dried milk containing a 1:5000 dilution of horseradish peroxidase-conjugated affinity-purified antibodies against the primary antibodies (Sigma), and washed in TBS. Blots were developed using an enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden). 

To prepare cytosolic and nuclear extracts, whole lenses were washed with ice-cold PBS and homogenized in a hypotonic buffer (10 mM HEPES [pH 7.8], 1 mM EDTA, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 10 μg/mL pepstatin) on ice (Dounce homogenizer, B pestle; Bellco Glass, Inc.,Vineland, NJ). Cellular fractionation for GR localization assays was performed according to a procedure reported previously. ²⁰ The homogenate (total tissue fraction) was centrifuged at 1500g for 10 minutes at 4°C to isolate the cytosolic fraction as the supernatant and the nuclear fraction as the pellet. The pellets were suspended in a hypotonic buffer (containing 25% glycerol, 20 mM NaCl) and were extracted in a hypotonic buffer (containing 25% glycerol, 0.5 M NaCl) for 1 hour on ice. The cytosolic and nuclear fractions were clarified by centrifugation at 14,000g for 60 minutes at 4°C, and the supernatants were collected. GRs were concentrated by immunoprecipitation using rabbit polyclonal anti-GR antibody (Santa Cruz Biotechnology), and mouse monoclonal anti-GR antibody (Santa Cruz Biotechnology) was used to probe Western blots. 

Results

Induction of PSO by Dexamethasone

A previous report, ^{4 21} showing that both the prednisolone concentration and the incidence of cataract formation is higher in the lenses of patients given prednisolone in eye drops than in those who receive it systemically, suggests that glucocorticoid affects lens pathogenesis directly. To examine the effects of glucocorticoid on the lens, we developed an ex vivo rat lens model. We isolated rat lenses and treated them in organ culture to cause steroid-induced cataracts directly. The lenses were incubated with 1, 3, and 5 μM dexamethasone and morphologic changes in the whole lenses were recorded photographically with a stereomicroscope. Lenses with posterior opacity were selected. The opacity first appeared at day 5 in the posterior region of lenses incubated with high concentrations (5 μM) of dexamethasone (data not shown). The opacity (Fig. 1D) in those lenses incubated with 5 μM dexamethasone for 7 days was more obvious, whereas it was not detected in the anterior or equatorial regions (Fig. 1C) . Untreated control lenses remained transparent until day 7. 

Histology of the Opaque Lenses and Localization of Dexamethasone

To characterize the effects of dexamethasone on cataract formation, organ-cultured rat lenses were examined histologically. The control lenses had normal cellular architecture. The epithelial cells maintained a monolayer, and the lens fiber cells were regularly packed (Figs. 2A 2C 2E) . Seven days after culture with dexamethasone, the architecture of the anterior region was relatively normal in the lenses in which opacities developed (Fig. 2B) . Distinct abnormalities were observed in the equatorial and posterior regions (Fig. 2D , Fig. 3B ). The opaque lenses exhibited elongated nuclei and irregular arrangements of lens fiber cells in the bow area of the equatorial region, compared with control lenses (Fig. 2D , arrowhead). Epithelial cells migrated along the lens capsule toward the posterior pole (Fig. 2D , arrows). Furthermore, circular lesions in the tissue appeared in the posterior regions of the opaque lenses. The posterior cortex under the posterior capsule had varying degrees of circular lesions (Fig. 3B , arrowhead). We used TEM to observe in more detail the morphologic changes at the cellular level. The fiber cells in the posterior lens displayed a disordered arrangement, and the tight contact between fiber cell membranes had separated (Fig. 3D) . Circular lesions were located between the lens fiber cells, or the fiber cell and capsules, but not intracellularly (Fig. 3D) . 

To determine whether the occurrence of opacity was due to an accumulation of dexamethasone, we investigated the localization of dexamethasone. Fluorescently labeled dexamethasone was added to the cultured rat lenses for 4 days, and the lenses were incubated for a further 3 days after the withdrawal of dexamethasone. The fluorescent label was observed in the posterior region where the opacity had developed (Fig. 4G) and in the equatorial region (Fig. 4H) . In contrast, fluorescence was not detected in the anterior region of the opaque lens (Fig. 4F) . These results suggest that the accumulation of dexamethasone has direct effects on the formation of opacity in the posterior region of the lens and on the abnormal migration of epithelial cells in the equatorial region. 

Effects of Dexamethasone on GSH Levels in Cultured Rat Lens

Investigators in previous studies have reported that glucocorticoid decreases the levels of GSH. Therefore, we tested whether dexamethasone reduces GSH levels. As shown in Figure 5 , comparison of GSH levels in rat lenses cultured for 5 or 7 days revealed no significant differences between control and dexamethasone-treated lenses. 

Expression and Localization of the Glucocorticoid Receptor

We confirmed the presence of the GR in the lens epithelium by using immunohistochemistry, and investigated GR expression in the opaque lenses induced by dexamethasone treatment. Immunohistochemical analysis of the lens epithelium revealed that GR in the normal lens was expressed in the epithelium of the equatorial region, whereas GR expression in the opaque lenses appeared in epithelial cells and in the abnormal cells that migrated sporadically toward the lower part of the equatorial line (Fig. 6A) . 

We examined the expression of the GR in the cytoplasmic and nuclear fractions to determine the subcellular localization of GR in the rat lens epithelium (Fig. 6B) . In nontreated lenses, most of the GR was observed in the cytoplasmic fractions (Fig. 6B , compare control lanes C and N). In contrast, in lenses in which opacity developed in response to dexamethasone, GR was significantly reduced in the cytoplasmic fractions and was observed in the nuclear fraction (Fig. 6B , Dexamethasone, lanes C and N). These results suggest that GR is translocated by dexamethasone from the cytoplasm into the nucleus in the rat lens epithelium. 

Dexamethasone Reduces Cadherin Protein Levels

Figures 2 and 3 show that the epithelial cells in dexamethasone-treated lenses migrated abnormally and that the fiber cell junctions were separated between the lens fiber cells, resulting in a disordered arrangement. We analyzed the expression of E- and N-cadherins to determine whether they were involved in these morphologic changes. No differences were observed in the levels of E- or N-cadherin mRNAs in the whole control lenses and the whole opaque lenses (Fig. 7A) . However, the E-cadherin protein levels were reduced in the opaque lens fractions compared with the control lenses, and N-cadherin was slightly reduced, whereas the levels of β-catenin were unaffected (Fig. 7B) . The most common mechanism by which GR is ligand activated is by binding to a glucocorticoid responsive element (GRE), which then regulates gene expression at the transcriptional level. However, the mRNA levels of cadherin were unchanged in the opaque lenses. This suggests that GR translocation is indirectly involved in the destabilization of cadherin protein. 

Effect of the GR Antagonist RU486 on Induction of Opacity by Dexamethasone

Our data show that dexamethasone was localized to the opaque region (Fig. 3C) , which suggests that accumulated dexamethasone can cause opacity by directly binding to lens proteins. Therefore, we used a GR antagonist, RU486, to determine whether the opacity is caused by GR-mediated signaling. The lenses were cultured in the presence of 5 μM dexamethasone, 5 μM RU486, dexamethasone and RU486 (Dex+RU486), or control medium. Treatment with RU486 reduced the number of lenses that had dexamethasone-induced opacification (Table 1) . Moreover, the opacity was dramatically reduced in the rat lenses incubated with dexamethasone plus RU486 compared with the lenses treated with dexamethasone only (Fig. 8A) . We also examined the effects of RU486 on expression of E-cadherin. As shown in Figure 8B , the reduction in E-cadherin protein levels induced by dexamethasone was blocked by treatment with RU486. 

Discussion

Glucocorticoid plays important regulatory roles in cellular physiology. Glucocorticoid freely penetrates the cell membrane and complexes with a specific receptor in the cytoplasm. This complex is translocated into nucleus. ^{22 23} GRs are expressed in the ocular tissue in the iris, ciliary body, cornea, sclera, trabecular meshwork, Schlemm’s canal, and lens. ²⁴ In this study, we used immunohistochemistry to show that the GR is expressed in rat lens epithelium, that most GR was localized in the cytoplasm, and that dexamethasone increased GR levels in the nucleus and decreased them in the cytoplasm. The reduced transfer of GR DNA-binding mutants to the nucleus after treatment with steroids ²⁵ shows that the translocation of GR is important for the response of the GR activity. Dexamethasone, a glucocorticoid, binds with high affinity to the GR, and has K _d in the 5- to 10-nM range. ^{26 27} Our data show that a high concentration (5 μM) of dexamethasone induced opacity in the posterior subcapsular region in organ-cultured rat lens, although this concentration was approximately 200- to 1000-fold higher than the K _d value of the drug. Previous studies have showed that 10 μM dexamethasone induces GR-mediated gene transcription and maximum GR translocation in a mouse fibroblast cell line. ^{28 29} This phenotype is morphologically similar to the lens opacity associated with human steroid-induced cataracts. ¹ Analysis of lens sections showed that opaque lenses exhibit abnormal changes in lens fiber cells, involving fiber cell separation, circular lesions, and irregular arrangements. These changes are similar to those described in rabbit lenses injected with prednisolone. ³ Therefore, these results suggest that the effects of dexamethasone on rat lens are probably mediated by binding the GR. 

Because the prednisolone degradation rate is much lower in the posterior cortex than in either the anterior cortex or the equator, steroid accumulation in the posterior region may directly induce PSC. ³⁰ To test whether the accumulation of dexamethasone influences the opacity in the posterior region, dexamethasone localization after withdrawal was investigated. Accumulated dexamethasone was unexpectedly observed in the equatorial region and also in the posterior region. The accumulated dexamethasone reduced the GSH level, which may directly induce opacity. ³¹ This hypothesis is supported by previous reports that the loss of GSH in lenses treated with dexamethasone was involved in cataractogenesis. ⁷ However, because GSH levels were not significantly changed in opaque rat lenses treated with dexamethasone, a reduction in GSH levels seems unlikely to be a major cause of opacity in our model. In this study, abnormal lens epithelial cells were identified in the equatorial region migrating along the capsule to the posterior region. This is interesting, because lens epithelial cells migrate from the equatorial region to below the posterior capsule in steroid-induced cataracts in human patients. ³² We did not detect abnormal epithelial cells in the opaque region of the cultured rat lens. This raises the question of whether the abnormal migrating cells directly develop into PSC. However, it is also possible that abnormal epithelial cell migration caused by glucocorticoid may lead to the disruption of normal epithelial fiber differentiation. At the transition zone, differentiation of the lens epithelial cells into fiber cells is responsible for the growth of the lens. The differentiated fiber cells elongate and attach to the posterior lens capsule. 

Cadherin binds directly to either β-catenin or γ-catenin (also called plakoglobin), and this complex is coupled to the cytoskeleton by α-catenin. ³³ This event is essential for cadherin participation in the formation of adherence junctions. ³⁴ E-cadherin, which acts to maintain the structure and function of the epithelium, ³⁵ is expressed in the epithelium of the lens. ³⁶ N-cadherin is a cell-cell adhesion molecule that is present in lens fibers. ³⁷ In this study, we found that a reduction in E-cadherin protein, which is associated with cell migration, ³⁸ was induced during the development of opacity. Furthermore, cadherins mediate cell-matrix adhesion, ³⁹ as well as cell-cell adhesion. Therefore, the downregulation of E-cadherin protein may have induced the abnormal migration of epithelial cells along the capsule. Because the abnormal cells may have been impeded in their differentiation into fiber cells, it is possible that the adhesion of fiber cells to the posterior capsule was obstructed or the interaction between fiber cells was disrupted. Therefore, the opacity may be caused by the abnormal migration of lens epithelial cells. To test this hypothesis, we analyzed the changes in E-cadherin protein expression and opacity formation after incubation with the glucocorticoid antagonist RU486. RU486 not only suppressed the formation of opacity but also inhibited the reduction in E-cadherin protein caused by dexamethasone. These results suggest that dysregulation of E-cadherin mediated by GR signaling is necessary for the formation of dexamethasone-induced cataracts. 

In summary, we have demonstrated that the development of opacity is induced by dexamethasone in the posterior subcapsular region of the rat lens and requires GR-mediated signaling. This opacity may proceed through the migration of abnormal epithelial cells, which results in the separation of lens fiber cells. Moreover, we found that the reduction in cadherin is regulated by dexamethasone in the rat lens. Although E- and N-cadherin protein levels were reduced by dexamethasone, it is still possible that the reduction in cadherin is the result of developing opacity. Whether the reduction of cadherin directly induces the cataract is currently under investigation. Future experiments will focus on the precise mechanisms underlying the GR-mediated reduction in cadherins by dexamethasone. 

 Supported by Korea Research Foundation Grant KRF-99-042-F00116.

 Submitted for publication June 16, 2002; revised November 20, 2002; accepted December 9, 2002.

 Disclosure: J. Lyu, None; J.-A. Kim, None; S.K. Chung, None; K.-S. Kim, None; C.-K. Joo, 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.

 

Effects of dexamethasone on lens transparency. Whole lenses were cultured for 7 days without (A, B) or with (C, D) dexamethasone. Photographs were taken at the focus of the posterior zone (B, D), or in the equatorial region (A, C) through the anterior pole. In the presence of dexamethasone, large posterior subcapsular opacities developed in the lenses. Without dexamethasone, lenses remained clear. Arrows: areas of opacity.

Figure 2.

 

Histology of lenses with dexamethasone-induced posterior opacity. Whole lenses were cultured for 7 days without (A, C) or with (B, D) dexamethasone. Sagittal sections were stained with DiOC₆ and ethidium bromide. The lens fiber cells of the equatorial bow region were disorganized and epithelial cells migrated along the lens capsule toward the posterior pole (D, arrow). The nuclei of lens fiber cells appeared elongated (D, arrowhead). In contrast, the anterior epithelium of the nontreated lens maintained a normal morphology (B). ep, epithelial cell; f, lens fiber. Scale bar, 20 μm.

Figure 3.

 

Morphologic changes in lens fiber cells within the opaque region. Semithin sections of the posterior region were stained with toluidine blue (A, B); an electron micrograph of the posterior region (C, D). At the posterior region of the opaque lens, a circular lesion (B, D, arrowheads) and irregular lens fiber cells (D) were present, and fiber interdigitations were disturbed (D, arrow). Clear lenses have normal fiber cells (A, C). f, lens fiber; c, lens capsule. Scale bar, (A, B) 20 μm; (C, D) 2 nm.

Figure 4.

 

Localization of dexamethasone in organ-cultured rat lens. Whole lenses were cultured with fluorescently labeled dexamethasone dissolved in DMSO (B, C, D, F, G, H) or with DMSO only (A, E). Photographs were taken at the focus of the anterior (B, F), posterior (C, G), and equatorial regions (D, H) through the anterior pole. Fluorescently labeled dexamethasone was localized within the dexamethasone-induced posterior subcapsular opacity (G) and within the postequatorial row (H), but not at the anterior pole (F). (A–D) Bright-field images; (E–H) fluorescence images. an, anterior; eq, equator; po, posterior.

Figure 5.

 

Effect of dexamethasone on GSH levels in the lens. Lens GSH concentrations were determined in rat lenses cultured for 5 or 7 days after dexamethasone treatment. Five lenses were used in each group to determine GSH levels, and experiments were performed in duplicate at least three times. Control (□); dexamethasone (▨). Data are expressed as the mean ± SEM.

Figure 6.

 

Immunostaining of flat lens preparations with anti-GR antibody and nucleocytoplasmic localization of GR after treatment with dexamethasone. (A) Flat preparations of equatorial zones in lenses cultured with (b) or without (a) dexamethasone, probed with anti-GR antibody. Antibody was detected with a rhodamine-conjugated secondary antibody. GR was strongly expressed in epithelial cells of the equatorial line and in migrating cells below the lens equator (b), but not in nontreated lenses (a). an, anterior; eq, equatorial line; po, posterior. Scale bar, 40 μm. (B) Lenses treated for 7 days with dexamethasone and control lenses, were fractionated to generate soluble cytosolic (C) and nuclear (N) fractions. These were analyzed by Western blots probed with an anti-GR polyclonal antibody, and horseradish peroxidase-conjugated secondary antibody. Anti-GR antibody recognized a prominent band at 95 kDa.

Figure 7.

 

Expression of E-cadherin, N-cadherin, and β-catenin in organ-cultured rat lens. (A) Transcripts of E-cadherin, N-cadherin, and β-catenin were analyzed by RT-PCR. Samples of total RNA (5 μg) were isolated from lenses that showed development of posterior opacity after treatment with dexamethasone and from clear lenses (C), and RT-PCR was performed. β-Actin was used as the control for protein loading. In the lenses with posterior subcapsular opacity, transcription of cadherin and β-catenin was unchanged compared with the control lenses. (B) Levels of E-cadherin, N-cadherin, and β-catenin were determined by Western blot analysis. Lenses that showed development of posterior opacity after treatment with dexamethasone (Dex) and clear lenses (C) were lysed and total proteins separated by SDS-PAGE. The associated proteins were subjected to Western blot analysis and probed with antibodies against E-cadherin, N-cadherin, and β-catenin.

Table 1.

 

Effects of Dexamethasone and the Inhibitor RU486 on Cultured Rat Lenses

Treatment (Number of Lenses)	No Opacities	Anterior	Posterior	Anterior + Posterior
Cont (41)	39 (95.1)	0	0	2 (4.9)
Dex (61)	5 (8.2)	6 (9.8)	37 (60.7)	13 (21.3)
RU486+Dex (47)	29 (61.7)	3 (6.4)	9 (19.1)	6 (12.8)
RU486 (46)	42 (91.3)	1 (2.2)	0	3 (6.5)

Figure 8.

 

The GR antagonist RU486 inhibited the development of dexamethasone-induced posterior subcapsular opacity. (A) Lenses were incubated for 7 days in the presence (+) and absence (−) of 5 μM dexamethasone (Dex) and 5 μM RU486, and whole lenses were photographed at the focus of the posterior level through the anterior pole. Lenses incubated in the presence of dexamethasone alone showed posterior subcapsular opacity (b), but lenses incubated with dexamethasone and RU486 (d), with RU486 only (c), or in the absence of dexamethasone (a) showed no opacity. (B) Equal amounts of protein lysates from lenses that were incubated for 7 days in the presence or absence of dexamethasone or with RU486 were separated by SDS-PAGE and blot. E-cadherin and actin were detected with specific antibodies. Actin was used to control for equal protein loading.

The authors thank Eak-hoon Jho, PhD, Dea-Myung Jue, PhD, and Jin Kim, PhD, for many helpful discussions during the research and Jang-hyun Kim for technical assistance.