Molecular Vision 2005; 11:853-858 <http://www.molvis.org/molvis/v11/a101/> Received 20 June 2005 | Accepted 30 September 2005 | Published 11 October 2005

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Expression of superoxide dismutase in whole lens prevents cataract formation

Dingbo Lin,¹ Micheal Barnett,¹ Laura Grauer,¹ Jerry Robben,¹ Annie Jewell,¹ Larry Takemoto,² Dolores J. Takemoto¹
 
 

¹Department of Biochemistry, ²Division of Biology, Kansas State University, Manhattan, KS

Correspondence to: Dolores J. Takemoto, PhD, Department of Biochemistry, Kansas State University, 104 Willard Hall, Manhattan, KS, 66506; Phone: (785) 532-7009; FAX: (785) 532-7278; email: dtak@ksu.edu

Abstract

Purpose: Oxidative damage is a major factor causing cataracts, which account for almost half of human blindness cases worldwide. In this study, we wished to determine if overexpression of superoxide dismutase (SOD) in intact lenses could prevent cataract formation induced by oxidative stress.

Methods: Fresh, intact lenses from 6-week-old male/female Sprague Dawley rats were incubated with plasmid DNA encoding the human SOD1 (Cu/Zn-SOD) gene at 37 °C in a CO₂ cell culture chamber with 95% air and 5% CO₂. SOD1 expression was determined by western blotting and SOD enzyme activity. Lenses with or without overexpression of SOD1 were treated with H₂O₂ and cataract formation was examined. SOD1 regulation of protein kinase Cγ (PKCγ) was determined by PKCγ enzyme activity assay. Intact lens gap junctions were determined by dye transfer assay.

Results: In the lens overexpression system, SOD1 cDNA was fused to EYFP to generate EYFP:SOD1 fusion proteins which allow detection from endogenous SOD1. Incubation of intact lenses with plasmid DNA produced EYFP:SOD1 fusion proteins as determined by western blot using anti-GFP or anti-SOD1 antibodies. This caused significant increases in SOD enzyme activity. Data indicated that SOD1 plasmid DNA can be expressed as a functional enzyme in intact lenses in culture. Lenses overexpressing SOD1 remained clear after H₂O₂ treatment at 100 μM for 24 h, similar to control. Overexpression of SOD1 diminished the effect of H₂O₂ on PKCγ activation and subsequent inhibition of gap junctions, indicating that overexpression of SOD1 may reduce reactive oxygen species (ROS) production, and this would prevent the normal H₂O₂ effect on cataract formation.

Conclusions: Overexpression of SOD1 in whole lens prevents H₂O₂-induced oxidative damage (cataract formation) to the lens and subsequent control of gap junctions by protein kinase Cγ.

Introduction

Cataracts account for approximately 50% of blindness worldwide [1]. Nearly one fourth of the cases of blindness in the United States are a result of cataracts. Oxidative damage is a major factor in both diabetic and age-related cataract formation [2,3]. Free radicals have been suggested to trigger cataract formation [4,5]. The toxic effects of these reactive oxygen species and free radicals can be eliminated by enzymes such as superoxide dismutase (SOD) which eliminates O₂^- to produce H₂O₂. This is then eliminated by glutathione peroxidase or by catalase.

Multiple SOD isoenzymes are present in human eyes, consisting of the extracellular SOD (EC-SOD), the cytosolic copper- and zinc-containing SOD (Cu/Zn-SOD, or SOD1), and the mitochondrial manganese-containing SOD (Mn-SOD) [6]. Cu/Zn-SOD (SOD1) is widely distributed and comprises up to 90% of the total SOD [7]. SOD1 is the most abundant SOD in the lens [3,7]. A loss or decrease in SOD enzymes can allow free radicals to induce irreversible effects and subsequent pathologies, such as Parkinson's disease, Alzheimer's disease, several neurological disorders, and cataracts [7-9]. SOD has been found to decrease in individuals with both senile and diabetic cataracts [10] and animal models with either SOD deficiencies or knock-outs show numerous ocular pathologies [11-14]. Cells with higher SOD enzyme activity are more resistant to the oxidative damage caused by H₂O₂, O₂^- and UV-B radiation [15]. Overexpression of SOD reduces oxidative damage and extends the life span in S. cereviseae and Drosophila [16], but the mechanism is not clear.

Since lens opacification (cataract) usually occurs over a long time period, development of a nonsurgical pharmacological treatment that can delay the opacification process would be beneficial. The lens epithelium is a nucleated cell layer that can be engineered to uptake or overexpress macromolecules which are known to protect the lens against oxidative damage [17]. In this study, we have demonstrated that overexpression of SOD1 cDNA in intact lenses in vitro could provide protection from H₂O₂-induced lens opacification. We have also determined that one mechanism by which SOD prevents H₂O₂-induced oxidative damage to the lens is through control of gap junctions by protein kinase Cγ. This would have high impact on the treatment of cataracts through the use of the lens as a "factory" to produce antioxidant enzymes which have been found to prevent oxidative damage and cataracts after H₂O₂.

Methods

Materials

Monoclonal antibodies against PKCγ and GFP were purchased from BD Biosciences (Palo Alto, CA). Rabbit polyclonal antibody against human SOD1 was purchased from Abcam (Cambridge, MA). Superoxide dismutase assay kit was purchased from Cayman Chemical (Ann Arbor, MI). Protein A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse IgG conjugated with HRP and PepTag nonradioactive protein kinase C assay system were purchased from Promega (Madison, WI). Dulbecco's Modified Eagle Medium (DMEM, low glucose), gentamicin, and penicillin/streptomycin were purchased from Invitrogen Life Technologies (Carlsbad, CA). H₂O₂, sodium fluoride (NaF), and EGTA were purchased from Fisher Scientific (Pittsburgh, PA). Na₃VO₄, phenylmethanesulfonyl fluoride (PMSF), Triton X-100, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO). Lucifer yellow, rhodamine dextran and SlowFade antifade were purchased from Molecular Probes (Eugene, OR).

Animals

Six-week-old male/female Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were sacrificed by CO₂. Enucleated lenses were used immediately in all assays. All experiments conformed to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and were performed under an institutionally approved animal protocol.

SOD1 construct and whole lens transfection

Human SOD1 cDNA was amplified by polymerase chain reaction followed by cloning into the BamHI-NheI sites of pEYFP-N1 (BDBiosciences Clontech, Palo Alto, Ca) as described [18]. The insertion was confirmed by sequencing. Fresh lenses were incubated in serum-free DMEM (low glucose) for 24 h at 37 °C with 5% CO₂. Clear lenses were incubated for an additional 24 h in 50 μg/ml SOD1 or pEYFP-N1 plasmid DNA followed by processing for subsequent assays. Expression of SOD1 was determined by western blot and SOD enzyme activity assays.

SOD enzyme activity assay

Whole lenses were homogenized and sonicated in 100 μl extract buffer. After centrifugation, the supernatants were used for testing according to the manufacturer's instruction. Enzyme activity was expressed as percent of control (untreated) specific SOD activity.

Western blot and immunoprecipitation

The whole lenses were homogenized with cell lysis buffer followed by sonication. The cell lysis buffer contained 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 0.1% protease inhibitor cocktail, 5 mM NaF, 5 μM Na₃VO₄, and 2 mM PMSF. After centrifugation at 12,000x g for 20 min, the supernatants were collected and used as whole cell extracts. Western blotting was carried out as described previously [19]. Whole cell extracts were immunoprecipitated with antisera at 4 °C for 4 h. Immunoprecipitation complexes were resolved by SDS-PAGE and protein patterns were visualized by western blot with particular antisera as indicated.

SOD prevents H₂O₂-induced cataract formation

Lenses overexpressing SOD1 or EYFP were treated with 100 μM H₂O₂ for 24 h at 37 °C in the CO₂ incubator. H₂O₂ was added into the media every 4 h for 6 times total. After 24 h, the lenses were washed with phosphate-buffered saline (PBS), and were photographed to examine cataract formation.

PKCγ activity assay

PKCγ activity was analyzed by use of the PepTag assay kit as described previously [20,21]. SOD1 transfected lenses with or without 24 h 100 μM H₂O₂ treatment were incubated in fresh DMEM serum free media after rinsing three times with PBS. After that, lenses were incubation with 100 μM H₂O₂ for 20 min, washed with PBS, and the supernatants of whole lens homogenates were collected. Equal protein amounts of whole lens cell extracts with or without H₂O₂ treatments were immunoprecipitated with PKCγ antisera (1:75) at 4 °C for 3 h. Immunoprecipitated PKCγ/agarose bead complexes were used for PKCγ enzyme sources. The reaction mixture contained 20 mM HEPES (pH 7.4), 1.3 mM CaCl₂, 1 mM DTT, 10 mM MgCl₂, 1 mM ATP, 0.2 mg/ml phosphatidylserine, and substrate PepTag peptides (PLSRTLSVAAK). The reactions were initiated at 30 °C for 25 min, and were stopped by placing the tubes in a boiling water bath for 10 min. Fluorescent PepTag peptides (PKCγ reaction products) were resolved by 0.8% agarose gel electrophoresis and visualized under UV light. The phosphorylated peptide bands were excised, and their fluorescence intensities were quantified by spectrophotometry at 570 nm. The enzyme activity was normalized by calibration of the relative level of phosphorylated substrates to the relative amount of PKCγ in the immunoprecipitation as determined by western blotting, and activity was expressed as percent of untreated specific PKCγ activity.

Gap junction activity-dye transfer assay

Whole lens gap junction activity was analyzed by dye transfer assay as described previously [22,23]. SOD1 transfected lenses were incubated with 100 μM H₂O₂ for 24 h. Lucifer yellow (2.5 mg/ml in PBS) was added to the lenses by microinjection as described [22,23], and the lenses were subsequently incubated in serum-free DMEM at room temperature for 30 min to allow dye transfer. Rhodamine dextran (1%) was used as a control for nonspecific leakage at the same time as the lucifer yellow dye. Lucifer yellow and rhodamine dextran were injected into the superficial cortical fibers per injection site using a microinjection apparatus (Nanoliter 2000; World Precision Instruments, Inc., Sarasota, FL). After incubation, the lenses were fixed in 2.5% paraformaldehyde, dissected, and mounted in 3% agar. The extent of dye transfer (μm, diffusion distance of rhodamine-dextran subtracted from lucifer yellow diffusion distance) as gap junction activity in the lens was determined by confocal microscopy. Each experimental group contained 6 lenses and distance of dye transfer was determined in 6 areas of the bow region of each lens using coded samples. Results are expressed as mean±SEM with p<0.05.

Statistical analysis

All analyses represent at least triplicate experiments. The statistical analysis employed in this paper is the Student's t-test. The level of significance (*) was considered at p<0.05. All data are mean±SEM.

Results

Expression of SOD1 in intact lenses in culture

In the fresh lens overexpression system, SOD1 cDNA was fused to EYFP to generate EYFP:SOD1 fusion proteins which are distinguished from endogenous SOD1. Expression specificity was confirmed by overexpression of EYFP tag only or by incubation without any plasmid DNA (PBS instead, designated as Control). Fresh lenses were incubated with plasmid DNA (50 μg/ml) for 24 h at 37 °C. The supernatants from whole lens extracts were used to determine the expression of SOD1 by western blotting using anti-GFP antibody (Figure 1A) or anti-human SOD1 antibody (Figure 1B). This anti-GFP antibody could recognize GFP, EGFP, EYFP, and DsRed serial proteins created by BD Biosciences Clontech (Palo Alto, CA). Western blot data shown in Figure 1A demonstrated that incubation of intact lenses with plasmid DNA produced protein expression of both single EYFP and SOD1:EYFP fusion proteins. No signal was detected in the lenses treated with phosphate-buffered saline (PBS). Figure 1B shows that EYFP:SOD1 fusion proteins were expressed with increases of 164% over control total SOD1 protein levels. Data indicates that SOD1 plasmid DNA can be uptaken and expressed specifically in the intact lenses in culture.

To further confirm if the overexpressed SOD1 has enzyme activity, SOD enzyme activity analysis was performed. Lenses incubated with or without plasmid DNA were harvested and SOD enzyme activity in the supernatants containing SOD1 was tested as shown in Figure 1C. Lenses incubated with PBS (control) or pEYFP-N1 empty vector plasmid DNA had very similar levels of SOD enzyme activity. However, incubation of lenses with SOD1:EYFP plasmid DNA caused significant increases in SOD enzyme activity (about 140% over control). Combined with western blot data, we concluded that SOD1 plasmid could be expressed as a functional SOD1 enzyme in the intact lenses in culture.

Effects of overexpression of SOD1 on cataract formation induced by H₂O₂

Previous reports show that cataract lenses have lower SOD levels than normal lenses [2,24-26]. We wished to determine if the increased SOD level in intact lenses in culture could prevent cataract formation induced by H₂O₂. SOD1 plasmid DNA-treated lenses (24 h) were exposed to 100 μM H₂O₂ for an additional 24 h. Lens opacification was monitored as shown in Figure 2A. In each treatment, six clear lenses with or without plasmid DNA treatments were used for H₂O₂-triggered cataract assay for an additional 24 h. Additional 24 h H₂O₂ incubations, every 4 h, or 6 times, with normal serum-free DMEM media caused one out of six lenses to become opaque. H₂O₂ for 24 h induced opacification in all six lenses. Lenses expressing EYFP protein had 3 opaque and 3 normal with H₂O₂. Lenses overexpressing SOD1 remained clear after H₂O₂ at 100 μM for 24 h, similar to control (Figure 2B). The results suggest that overexpression of SOD1 prevents lens cataract formation triggered by H₂O₂.

Effect of overexpression of SOD1 on PKCγ activation by H₂O₂

The lens is an avascular tissue that can maintain homeostasis without a vascular system. Control of gap junctions by PKCγ is very important to prevent lens cells from oxidative damage [21]. We wished to determine if overexpression of SOD1 would alter lens gap junctional communication by PKCγ by elimination of ROS. This, in turn, could result in abolished effects of H₂O₂ on cataract formation.

H₂O₂ activation of PKCγ was determined at two time points, before and after long-term (24 h) H₂O₂ treatments. First, we determined PKCγ enzyme activity in the lenses transfected with or without SOD1 before long-term (24 h) H₂O₂ treatment. Lenses were incubated with 100 μM H₂O₂ at 37 °C for 20 min. PKCγ enzyme activity analysis was done as shown in Figure 3 (before 24 h H₂O₂). H₂O₂ activated PKCγ enzyme activity in whole lenses with or without EYFP expression, consistent with our previous report [20]. In contrast, in the lenses overexpressing SOD1 PKCγ was not activated by H₂O₂ although PKCγ levels were slightly increased before H₂O₂ treatment for 20 min. After 24 h H₂O₂ treatment, PKCγ enzyme activities in lenses without overexpressed SOD1 were at very low basal levels (Figure 3; control and pEYFP), and they were not activated by addition of H₂O₂. However, lenses with SOD1 overexpression still had similar basal levels of PKCγ enzyme activity compared to the control. Addition of H₂O₂ did not cause increases of endogenous PKCγ activity (Figure 3; after 24 h H₂O₂). Long-term H₂O₂ (24 h) caused decreases in PKCγ protein levels (data not shown), most likely due to persistent activation, a process known to cause PKC degradation. These results indicate that overexpression of SOD1 may reduce reactive oxygen species (ROS) production, and this would prevent the normal H₂O₂ activation of PKCγ.

Effect of overexpression of SOD1 on gap junctions in intact lens

Activation of PKCγ results in an inhibition of gap junctions in lens epithelial cells and whole lenses in culture [21,23]. Gap junction dye transfer was significantly decreased in H₂O₂ treated lenses without SOD1 overexpression (Figure 4). In contrast, overexpression of SOD1 slightly increased and H₂O₂ did not decrease gap junction dye transfer in the SOD1 overexpressing lenses. The slight increase in dye transfer in the SOD1 overexpressing lenses without H₂O₂ may reflect a lowering of endogenous ROS and subsequent return of PKCγ to complete basal levels. This would be reflected in a greater dye transfer activity.

Discussion

ROS and SOD have been implicated in many disease states including cataract. Yet, SOD has not been previously used in cataract treatment. In this study, we have demonstrated that application of SOD1 plasmid DNA directly on intact lens produces enzymatically active SOD1 proteins (Figure 1). Of interest, overexpression of SOD1 prevents cataract formation in the lenses (Figure 2). We have also determined that one mechanism by which SOD prevents H₂O₂-induced oxidative damage to the lens is through control of gap junctions by protein kinase Cγ.

Translocation of macromolecules into whole lenses has been reported previously [17,27-29], but very little is known about the mechanism. The generally accepted explanation is that macromolecules such as proteins can pass through the anterior capsule or capsule at the equatorial regions, and are then endocytosed by epithelial cells and/or outer cortical fiber cells [17]. In this study we have, for the first time, reported that plasmid DNA such as SOD1 can pass into the whole lenses without any carrier, and SOD1 proteins can be translated with normal enzyme activity (Figure 1). Functional SOD could eliminate cellular ROS such as superoxide anion (O₂^-) and generate H₂O₂ which is then eliminated by catalase. High ROS and free radicals trigger lens cataract formation [4,5]. Application of H₂O₂ may cause cellular damage which subsequently results in cataract formation (Figure 2; control and pEYFP columns). However, in the intact lenses with overexpressed SOD1, H₂O₂-induced cataract was diminished to the control levels (Figure 2; pEYFP:SOD1 column). This may be due to increased SOD elimination of cellular ROS produced by application of H₂O₂. The elevated SOD enzyme appears to act as a protection against oxidative stress induced by H₂O₂. Increased SOD1 levels in the overexpressing intact lenses may maintain much lower ROS levels than those of normal intact lenses in culture which, in turn, lead to increasing lens clarity. Therefore, we conclude that overexpression of SOD1 eliminates cellular ROS triggered by addition of H₂O₂ which subsequently prevent cellular oxidative damage and cataract formation.

Gap junctions are very critical to lens homeostasis. Mutations in Cx43, Cx46, and Cx50 have been linked to cataracts [30-33]. We have previously shown that PKCγ is a predominant PKC which can be activated by H₂O₂ in the lens via an oxidation mechanism [21]. Activated PKCγ phosphorylates Cx43 and Cx50 which results in a closure of gap junctions in the lens when exposed to stress [20,21,23]. This may provide a protective effect on the intact lens against stress damage. In this current study, short term H₂O₂ treatment (20 min) stimulated significant cellular PKCγ activation, consistent with our previous reports in lens epithelial cells [21] or in intact lenses [20], suggestive of PKCγ activation via an oxidation mechanism [21]. However, H₂O₂ activation of PKCγ was completely diminished in the intact lenses with overexpression of the human SOD1 gene (Figure 3), indicating that elevated SOD removed extra ROS generated by H₂O₂ and maintained cellular ROS at a low/physiological level which keeps cellular PKCγ in an inactive form (Figure 3; pEYFP:SOD1 columns).

Prolonged oxidative stress (such as long term H₂O₂ treatment for 24 h) causes protein oxidation, aggregation, degeneration, and cell death. This subsequently resulted in cataract formation as shown in Figure 2. Prolonged H₂O₂ treatment in the lens may trigger cell apoptosis which subsequently causes lens cataract. This may account for the decreases in gap junction dye transfer (Figure 4; control with H₂O₂ 24 h). Overexpression of SOD1 could remove cellular ROS which subsequently abolishes this H₂O₂ effect and physiologically maintain PKCγ inactive (Figure 3) and gap junctions active (Figure 4; pEYFP:SOD1 before and after H₂O₂) which are critical to maintain lens homeostasis when lenses were exposed to H₂O₂ stress. This was also reflected by cataract formation experiments (Figure 2). We conclude that overexpression of SOD1 may prevent H₂O₂-induced oxidative damage to the lens partially through control of gap junctions by protein kinase Cγ. The outcome of this study may help to develop gene therapies for cataractogenesis in the future.

Acknowledgements

We thank Dr. Roos of the University of Chicago for the human SOD1 plasmid. The research was supported by grants from the National Eye Institute to DJT (EY13421, EY15670) and to LT (EY02932) and from the National Organization for Rare Disorders (NORD) to DL. This is a publication 05-332-J from Kansas Agricultural Experiment Station, Manhattan, KS.

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