Induction of Thioltransferase and Thioredoxin/Thioredoxin Reductase Systems in Cultured Porcine Lenses under Oxidative Stress

Abstract

purpose. In view of the important antioxidant roles of thioltransferase (TTase), thioredoxin (Trx), and thioredoxin reductase (TR) in the lens, the present study was conducted to investigate the induction of these cytosolic enzymes in response to H₂O₂ stress in cultured lenses.

methods. Porcine lenses were cultured, exposed to H₂O₂ for various lengths of time between 0 and 24 hours, and photographed to detect morphologic changes. The lenses were then harvested; dissected into epithelial layer, cortex, and nucleus; and homogenized for the determination of the glutathione (GSH) level. Pooled epithelial layers were used to examine TTase, Trx, and TR protein or mRNA levels.

results. Treatment of lenses with H₂O₂ caused distinct morphologic changes. Lower concentrations of H₂O₂ (0.2 mM) caused the lens to be hazy after 6 hours and to worsen progressively between 12 and 24 hours. Higher levels of H₂O₂ (0.5 mM) induced similar morphologic changes, but sooner (within 1 hour) and more severe. Both H₂O₂-treated groups showed a dramatic and gradual GSH depletion during the 24-hour incubation, but the GSH level at 50% or above appeared to be essential in maintaining lens clarity. However, TTase, Trx, and TR activities, protein expressions, and mRNA transcriptions in the epithelial layers of these lenses were increased, but each enzyme had a distinct pattern. Under mild H₂O₂ stress, a slow and transient activation of TTase, Trx, and TR was observed. However, under stronger H₂O₂ stress, all three enzymes showed a very rapid increase and then a steady decline in activity. Western blot and RT-PCR analyses revealed that this increase in activity in all three enzymes was due to the induction of protein and mRNA expression. In the control group (no oxidative stress) all three enzyme activities and their respective expressions remained constant throughout the experimental period.

conclusions. The data show that TTase, Trx, and TR activity and expression are induced in lens cells under oxidative stress, probably to protect and maintain the health of the lens.

Oxidative stress induced by reactive oxygen species (ROS) has long been implicated in senile cataract formation. ^{1 2 3} ROS molecules are generated endogenously by enzyme systems in the lens, as by-products of normal metabolic processes, or exogenously from the environment. The main ROS molecules that generate in the lens include superoxide anion, hydroxyl radical, and H₂O₂. ² Endogenous systems that can generate ROS in the lens include NADPH oxidase, mitochondria, and peroxisomes (Rao PV et al. IOVS 1999;40:ARVO Abstract 907 and Wang Y, Lou MF. IOVS 2005;46:ARVO Abstract 1896). Two main sources that produce exogenous ROS are UV light and ionic radiation. ROS molecules produced through these processes in the lens are neutralized by antioxidants and oxidation defense systems. ^{2 3} Even in the presence of these powerful antioxidants and ROS-neutralizing enzyme systems, some ROS molecules get through these defense systems and oxidatively damage cellular molecules such as proteins, lipids, and DNA. ^{1 4} Hence, the lens is also equipped with enzyme systems that can repair the damaged molecules ¹ or degrade the severely damaged ones. ^{5 6} Thioredoxin (Trx)/thioredoxin reductase (TR) and thioltransferase (TTase)/glutathione reductase (GR) systems have been identified as powerful protein repair systems in mammalian tissues. ^{7 8 9} Recent work has shown that both TTase/GR and Trx/TR systems are present in the lens and play a critical role in maintaining the lens in a reduced state. ^{10 11}  

Trx is a small 12-kDa heat-stable protein. It is ubiquitously present in high and low forms of life. Its active site has two highly conserved vicinal cysteine residues that can reduce protein–protein disulfides. ¹² Oxidized Trx is reduced by TR and reduced nicotinamide adenine dinucleotide phosphate (NADPH). TR is a homodimeric selenoenzyme that catalyzes the NADPH-dependent reduction of Trx as well as numerous other oxidized cellular molecules. ¹³ The Trx/TR system plays an important role in the redox regulation of multiple intracellular processes, including DNA synthesis; cell proliferation, growth, and differentiation; protein regeneration; and resistance to cytotoxic agents that induce oxidative stress and apoptosis. ^{14 15 16}  

TTase is an 11.8-kDa heat-stable protein present ubiquitously in prokaryotes and eukaryotes. ¹⁷ It is also a multifunctional enzyme implicated to have a role in many biochemical processes such as protein regeneration, ⁸ reduction of ribonucleotide reductase, ¹⁶ catalysis of the dethiolation of protein-thiol mixed disulfide, ^{8 18} and reactivation of key glycolytic and oxidation defense enzymes. ¹⁹ In recent years, TTase has also been found to possess dehydroascorbate reductase activity ^{20 21} and to participate in the regulation of signal transduction. ²²  

Stress conditions, including oxidative stress, induce expression of many proteins and enzymes in various cell types. It has been shown that TTase expression is induced two- to threefold in human lens epithelial cells in response to a bolus of mild H₂O₂ stress. ²³ This TTase upregulation is mediated through redox signaling. ²⁴ In a similar fashion, Trx is also upregulated in human lens epithelial cells under the same oxidative stress conditions. ¹⁰ Trx expression has also been induced in Emory mouse lens exposed to photo-oxidation. ²⁵ However, it is not very well understood how TTase, Trx, and TR are upregulated in the lens in response to a long duration of oxidative stress, during which time lens opacity can be gradually emerged. We have undertaken this study to investigate the upregulation or induction of these two important oxidation damage repair systems in cultured porcine lenses under hydrogen peroxide stress conditions and have provided evidence that expressions of TTase, Trx, and TR are all upregulated, each with a distinct pattern. 

Materials and Methods

Fresh eyes from 6- to 8-month-old pigs were obtained from Farmland, Inc. (Crete, NE). Each lens was surgically removed from the eyeball through the anterior side 2 to 3 hours after death. Goat anti-human Trx-1 and TR-1 antibodies were purchased from American Diagnostic Inc. (Greenwich, CT). All the other chemicals and reagents were standard commercial products of analytical grade. 

Lens Organ Culture and Oxidation Exposure

Fresh porcine lenses were removed from the eyeballs and preincubated in TC-199 medium for 2 hours in a CO₂ incubator. Lenses were then transferred to 12-well plates containing 4 mL of fresh TC-199 medium and incubated for 24 hours. The lenses were divided into three groups, each with three lenses. One group was incubated without H₂O₂ in the medium. The second group was incubated in the medium containing 0.1 mM H₂O₂ and 2.31 units of glucose oxidase (GO) to achieve a final and constant concentration of 0.2 mM H₂O₂. To the remaining one third of the wells, 0.75 mM H₂O₂ and 4.63 units of GO were added to obtain a final and constant concentration of 0.5 mM H₂O₂. Addition of appropriate amounts of GO was to maintain a steady level of H₂O₂ during the course of this 24-hour-incubation. Fifty microliters of the medium was removed at 0-, 1-, 2-, 4-, 6-, 9-, 12-, 18-, and 24-hour intervals and H₂O₂ concentration in the medium was determined as described later in the article. Lens images were captured on culture plates at 0, 2, 6, 12, and 24 hours after H₂O₂ treatments (Fotovix system; Tamron Ltd., Saitama, Japan; coupled to a Snappy Frame Grabber; Play, Inc. [no longer in existence]). 

Preparation of Porcine Lens Homogenate

At the end of the desired incubation period, each lens was rinsed with PBS solution and blotted with a filter paper. The lens capsule epithelial layer was peeled off and saved, and the decapsulated lenses were discarded. Three epithelial layers were pooled and homogenized in 200 μL ice-cold buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol, 1 μg/mL aprotinin, and 100 μg/mL phenylmethylsulfonyl fluoride (PMSF) using a glass-to-glass homogenizer (Duall; Kontes Glass Co, Vineland, NJ). The homogenate was centrifuged at 13,000g for 25 minutes at 4°C, and the supernatant was used for determination of enzyme activities and immunoblot analysis. For glutathione (GSH) measurement, the pooled epithelial layers were homogenized in the same buffer solution without EDTA and β-mercaptoethanol. 

For measurement of TTase distribution in the lens, the epithelial layers from three fresh lenses were removed and pooled. The decapsulated lenses were further dissected with surgical forceps and spatula into outer cortex, inner cortex, and nucleus at a 1:1:1 (wt/wt/wt) ratio and pooled, respectively. Each pooled lens portion was homogenized by a glass homogenizer in 200 (epithelium) or 500 (cortex or nucleus) μL buffer as just described. 

Preparation of Anti-TTase Antibody

Affinity-purified rabbit anti-TTase antibody was prepared using purified recombinant human TTase (or glutaredoxin-1) by Bethyl Laboratories, Inc. (Montgomery, TX). Antiserum was made by immunization of an albino rabbit. For primary immunization, 1 mg of recombinant human TTase was emulsified with Freund’s complete adjuvant, and injected subcutaneously. Eight weeks later, the a booster injection of 1 mg recombinant human TTase emulsified with Freund’s incomplete adjuvant was administered, and antiserum was collected 10 days after the booster. Antiserum was affinity purified with a TTase affinity column, which was prepared by linking TTase to agarose by the cyanogen bromide method. 

Purification of Porcine Liver TR

Porcine liver TR was purified according to the method of Arner et al., ²⁶ with modifications. The method includes several column chromatographic steps, including anion exchange by diethylaminoethyl (DEAE)-Sepharose, affinity by adenosine diphosphate (ADP)-Sepharose, anion exchange by Q-Sepharose, and gel filtration by Sephadex G-75. The enzyme was electrophoretically pure after these steps. 

Enzyme Assays

TTase activity was assayed according to a previously described method. ²⁷ Protein concentrations in tissue homogenates were determined by the bicinchoninic acid (BCA) method according to the manufacturer’s protocol (Pierce Biotechnology, Rockford, IL) with bovine serum albumin as the standard. TR activity was determined by DTNB (5,5′-dithiobis-(2-nitrobenzoic acid) assay, as described by Holmgren and Bjornstedt. ²⁸ The activity of Trx was determined by a previously described method. ^{11 28} This assay is based on the insulin reduction ability of Trx with NADPH in the presence of excess TR. Briefly, aliquots of Trx were preincubated at 37°C for 15 minutes with 2 μL of buffer containing 50 mM HEPES (pH 7.6), 100 μg/mL BSA, and 2 mM dithiothreitol (DTT) in a total volume of 70 μL. Then, 40 μL of a reaction mixture composed of 250 mM HEPES (pH 7.6), 2.4 mM NADPH, and 6.4 mg/mL insulin was added. The reaction was started by adding 10 μL TR (4.75 A₄₁₂ U/mL), and incubation was continued at 37°C for 20 minutes and terminated by adding 0.5 mL of 6 M guanidine-HCl and 1 mM DTNB, and the absorbance at 412 nm was measured. The rate of DTNB reduction was calculated from the increase in A₄₁₂, using a molar extinction coefficient of 27,200 M⁻¹ · cm⁻¹ (reduction of DTNB by 1 mole of Trx-(SH)₂ yields 2 moles of TNB, with a molar extinction coefficient of 13,600 M⁻¹ · cm⁻¹). One unit of activity was calculated by A₄₁₂ × 0.62 /13.6 × 2 as the micromoles of NADPH oxidized, because 1 mole of NADPH corresponds to 2 moles of the sulfhydryl group. 

GSH and H₂O₂ Assays

GSH concentrations in lens tissue homogenates were determined by the previously described method of Ellman. ²⁹ Hydrogen peroxide concentration in lens organ culture medium was measured by the method of Hildebrant et al. ³⁰  

Extraction of Total RNA and Detection of mRNA for TTase, Trx, and TR by RT-PCR

Porcine lens capsule epithelial layers were peeled off as described earlier and immediately transferred to an RNA stabilization reagent (RNAlater; Qiagen, Inc., Valencia, CA) and kept submerged at room temperature for 6 hours. After this tissue-stabilization step, total mRNA was extracted from the epithelial layers with a RNeasy mini kit (Qiagen), according to the manufacturer’s protocol. Aliquots containing equal amounts (1 μg) of mRNA were reverse transcribed with a cloned AMV first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). The following primers were designed and synthesized to detect TTase, Trx, TR, and β-actin cDNA. TTase, (forward) 5′-CCTGTCAGCATGGCTCAAGCATTT-3′, (reverse) 5′-ATCCACCAGGAAGCGCTGTCATTA-3′; Trx, (forward) 5′-GCTGCCAAGATGGTGAAGCAGATT-3′, (reverse) 5′-GCAACATCCTGACAGTCATCCACA-3′; TR, (forward) 5′-GCTTTGGAGTGCGCTGGATTTCTT-3′, (reverse) 5′-CGTGAAAGCCCACAACACGTTCAT-3′; β-actin, (forward) 5′-GTGGGGCGCCCAGGCACCA-3′, (reverse) 5′-CTCCTTAATGTCACGCAGGATTTC-3′. The sizes of the amplification products of TTase, Trx, TR, and β-actin were 390, 206, 715, and 420 bp, respectively. Equal amounts of synthesized cDNA were amplified by PCR with Taq DNA polymerase (Invitrogen), to detect mRNA for TTase, Trx, TR, and β-actin. The conditions used for PCR were 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute for 35 cycles. Aliquots were taken from PCR mixtures and analyzed by 1% agarose gel electrophoresis. 

Western Blot Analysis

Tissue homogenates were separated by 12% SDS-PAGE and transferred to a membrane (TransBlot; Bio-Rad, Hercules, CA). The membranes were probed with anti-Trx, anti-TR, or anti-TTase antibodies diluted in TBST buffer (10 mM [pH 7.5] Tris-HCl, 100 mM NaCl and 0.1% Tween-20) and then treated with either donkey anti-goat IgG-horseradish peroxidase (for membrane probed with goat anti-human Trx and TR antibodies) or goat anti-rabbit IgG-horseradish peroxidase (for membranes probed with rabbit anti-human TTase antibody) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunodetection was performed with chemiluminescent reagent (Santa Cruz Biotechnology). The immunoblot was analyzed with an imaging system (Fluor-S MAX MultImager; Bio-Rad). 

Results

Maintaining 0.2 and 0.5 mM H₂O₂ Concentrations in Lens Culture Medium

Cultured lens has an inherent ability to detoxify H₂O₂ in the culture medium within a very short time. When porcine lenses were incubated in TC-199 medium containing 0.2 or 0.5 mM H₂O₂, within 10 to 15 minutes, H₂O₂ in the medium disappeared. Therefore, an appropriate amount of GO was needed to maintain the desired H₂O₂ level in the culture medium during the 24-hour incubation period. Our results show that a near constant level of oxidative stress of 0.2 mM H₂O₂ and 0.5 mM H₂O₂ was maintained with combinations of 2.31 units of GO with 0.1 mM H₂O₂-containing medium and 4.63 units of GO with 0.75 mM H₂O₂-containing medium, respectively (Fig. 1inset). 

Effect of H₂O₂ on GSH Levels in Epithelial Layers of Cultured Lenses

Figure 1shows the GSH concentration in three pooled lens epithelial layers of each group at various time points. For an unknown reason, the GSH level in the control group dropped continuously in the first 4 hours and was maintained at 75% of the original value during the remaining 24-hour incubation period. Lenses incubated with 0.2 mM H₂O₂ showed a continuous decline in GSH up to 9 hours, with only 35% of the GSH retained. The depleted level remained constant between 9 and 24 hours of incubation. In lenses exposed to 0.5 mM H₂O₂, the GSH level dropped precipitously, and the depletion began sooner. GSH concentration was only 41% at 1 hour and further declined to 21% at 9 hours, in comparison with the basal level. This minimal amount of GSH was maintained until the end of the experiment. 

Morphologic Changes in Lenses under H₂O₂ Stress

The spontaneous loss of epithelial GSH (25%) did not affect the morphology of the lens, as the lenses of the control group maintained their transparency throughout the 24-hour experimental period (Fig. 2) . Lenses exposed to 0.2 mM H₂O₂ however, maintain clarity in the first 6 hours (Fig. 2)and then developed opacity at the anterior and posterior cortical regions with the effected area expanded with time. A higher H₂O₂ level induced opacity sooner, starting from 1 hour of incubation. Haziness in these lenses started from the anterior and posterior cortical regions and extended to the entire cortex as the exposure time was prolonged. However, the nuclear regions of these lenses remained clear at the end of 24 hours Comparison of the lenses in the two H₂O₂-treated groups at various time points showed that cataract formation was directly proportional to the concentration of H₂O₂ and the exposure time used in the experiment (Fig. 2) . 

Distribution of TTase Activity in Various Regions of Porcine Lens

The distribution of TTase activity was examined in various parts of the porcine lens. As shown in Figure 3 , the epithelial layer had the highest TTase activity, with nearly 84% of the total. The inner and outer cortex had approximately 14% of the total activity, whereas the nucleus had only 2% of the total. Because TTase activity was mainly concentrated in epithelial layer, the following experiments were conducted only on the epithelial layers. 

The Effect of H₂O₂ on TTase Activity and Expression in Cultured Lens

TTase activity stayed unchanged in control lenses incubated in the culture medium alone (Fig. 4A) . However, lenses incubated in 0.2 mM H₂O₂ showed a slow increase in TTase activity in which the activity peaked at 9 hours and then started to decline gradually. After 24 hours of post-H₂O₂ stress, TTase activity was still four times higher than that of the control. There was a more rapid increase in TTase activity in the 0.5-mM H₂O₂ group (Fig. 4A) , which peaked at 4 hours, with TTase activity 10 times higher than in the control at the same time point. After 4 hours, TTase activity started to decline gradually but even after 24 hours, the activity was still two times higher than in the control group. 

There was no significant difference in TTase expression in untreated lenses with time (Fig. 4B) , but we observed a gradual increase in TTase expression in lenses treated with 0.2 mM H₂O₂ (Fig. 4B) . This increase reached its peak at 9 to 12 hours and then gradually declined but was still above the basal level at the end of 24 hours. The lenses exposed to a higher level of H₂O₂ showed a rapid TTase expression compared with the 0.2-mM H₂O₂–treated group (Fig. 4B) . This increase in TTase expression reached its maximum at 2 to 4 hours and then started to decline but remained above the basal level at the end of the experiment. Figure 4Bshows the GAPDH expression as an internal control, to confirm that all samples used in this experiment contained equal amounts of total protein. 

Effect of H₂O₂ on Trx Activity and Expression in Cultured Lens

During the 24-hour experimental period, the control lenses showed no change in Trx activity (Fig. 5A) . However, Trx activity in lenses treated with 0.2 mM H₂O₂ had a very slow but steady increase, which reached its maximum (approximately threefold increase) over the control at 12 hours, before gradually returning to its basal level at 24 hours. Lenses treated with a high concentration of H₂O₂ had a rapid and transient increase in Trx activity that peaked at 2 hours after treatment and returned to the control level within 12 hours (Fig. 5A) . 

Immunoblot studies of Trx gene expression showed no change in control lenses (Fig. 5B) . However, there was a slow increase in Trx gene expression in 0.2-mM H₂O₂–treated lenses where the peak increase can be seen at 12 hours followed by a basal level within the next 12 hours. Lenses treated with 0.5 mM H₂O₂ showed a rapid increase in Trx expression (Fig. 5B)that continued up to 4 hours and returned to basal level by 12 to 24 hours. Again, the steady GAPDH expression indicated an equal amount of proteins used in this analysis. 

Effect of H₂O₂ on TR Activity and Expression in Cultured Lens

Similar to Trx and TTase, the TR activity of control lenses was not changed during the entire experimental period (Fig. 6A) . Lenses incubated with 0.2 mM H₂O₂ had a transient increase in TR activity. The peak in this increase (a twofold increase) was reached at 4 hours and then the activity returned to its original level within 12 hours. TR activity in lenses treated with 0.5 mM H₂O₂ showed a very rapid and transient increase that peaked (2.5-fold increase) at 1 hour and then returned to basal level at 4 hours (Fig. 6A) . After 4 hours, the level started to decline slightly, and by 24 hours it had returned to the basal level. 

TR expression in control lenses remained constant in this experiment (Fig. 6B) . However TR expression increased with time in 0.2-mM H₂O₂–treated lenses up to 4 hours (Fig. 6B) , after which a gradual decline in TR expression was observed. By 12 hours, it reached the basal expression level. Lenses treated with 0.5 mM H₂O₂ showed a rapid increase in TR expression, which peaked at 1 to 2 hours, returned to basal level within 4 hours, and maintained this level until the end of experiment. Figure 6Balso shows a constant GAPDH expression in these samples. 

Effect of H₂O₂ on TTase, Trx, and TR mRNA Expression

The organ culture conditions did not alter mRNA levels of TTase, Trx, or TR in control lenses without the presence of oxidative stress during the 24-hour experimental period (Fig. 7) . However, mRNAs in 0.5-mM H₂O₂–treated lenses for all three proteins were transiently upregulated (Fig. 7) . The patterns of mRNA upregulation for TTase, Trx, and TR correlated with the patterns of protein upregulation and activity increase for each respective enzyme. In the case of TTase, exposure to 0.5 mM H₂O₂ stimulated a rapid TTase mRNA upregulation starting at 1 hour and reaching its maximum by 4 hours before declining and returning to normal level by 6 hours. Similar rapid, abrupt upregulation of mRNAs for Trx and TR was also seen (Fig. 7)in lenses exposed to 0.5 mM H₂O₂. The stimulated TR mRNA upregulation appeared to last longer (up to 9 hours of incubation) before returning to the basal level (Fig. 7) . 

Discussion

The observation of spontaneous loss in lens epithelial GSH level under organ culture condition is in accord with the results of Qin et al. ³¹ and Wang et al. ¹⁸ , who found loss of GSH in cultured young rat and porcine whole lenses, respectively. The reason for such loss is not clear. Qin et al. ³¹ had thoroughly investigated various possibilities to prevent GSH depletion during culture conditions but such attempts were not successful, and the results were not conclusive. Exposing the lens to oxidative stress further depleted the GSH pool in the epithelium, but it appears that the lens was able to maintain its transparency as long as the GSH concentration was approximately 50% of the basal level (see Figs. 1 2 ). This critical point of lenticular GSH level for maintaining lens clarity has also been observed in other laboratories. ^{31 32} Even though we reported only the epithelial GSH levels and how they were affected by oxidative stress in this study, because the epithelial layer contains the highest density of GSH in the lens, alterations in the epithelial GSH pool may project the general redox status of the whole lens. 

Similar to the results of our previous study on TTase distribution in rat ¹⁰ and human ¹¹ lenses, the porcine lens also showed over 80% of its TTase activity concentrated in the epithelium. This may be because the lens epithelium is known to be constantly exposed to oxidant stress through direct photo-oxidation and oxidants from the aqueous humor. ³³ However, the presence of TTase in the cortex and nucleus indicates that TTase may play a physiological role in the differentiated fiber cells. Further studies are needed in these areas. 

Of interest, the transient upregulation patterns of enzyme activity and protein expression were similar for both TTase and Trx under high or low oxidative stress conditions (Figs. 4A 4B 5A 5B) . It is reasonable to expect that the lens has enough oxidation defense ability at initial stages to eliminate H₂O₂ and to reduce modified thiols in proteins and enzymes with the existing TTase and Trx; thus, the lens does not have to induce the expression of these two genes. However, under prolonged oxidative stress conditions the lens may reach a point where the intrinsic TTase and Trx are no longer capable of maintaining the redox homeostasis of the lens. Under such circumstances, the lens may begin to increase the gene expression to compensate for the increased workload. For the low stress condition, the critical time is approximately 6 hours. For the higher level of oxidative stress, this time point is shortened to 2 hours. In both cases, a transient upregulation of TTase and Trx occurred gradually and lasted 2 hours or more. The faster response and shorter upregulation period in lenses exposed to higher oxidative stress correlated with the faster morphologic changes in these lenses when oxidative stress is expected to overwhelm the defense mechanisms in the tissue. The results of transient upregulation of TTase and Trx in the porcine lens epithelium resemble that of cultured human lens epithelial (HLE) cells under mild oxidative stress conditions reported previously from our laboratory. ^{11 34}  

It is very intriguing to observe that the lenticular TR induction under the same experimental conditions was much faster than that of TTase and Trx. It is likely that this selenoprotein is an oxidation sensor, ³⁵ the faster induction of which would provide adequate reducing power to maintain Trx in the reduced state. Thus, Trx in turn can reduce the oxidized target proteins and enzymes to keep the redox balance within the cells. 

It has been shown that the expression of multiple genes of oxidative stress defense systems are controlled by the AP-1 transcription factor. ^{36 37} These genes include GR, ³⁸ glutathione S-transferase, ³⁹ Trx, ⁴⁰ and TR. ⁴¹ Li and Spector ⁴² and Li et al. ⁴³ have also shown that H₂O₂ mediates induction of proto-oncogenes. c-Jun, c-fos, and c-myc are controlled by AP-1 transcription factor in rabbit lens epithelial cells. Krysan and Lou ²⁴ have demonstrated in HLE B3 cells that the activation of the TTase gene under oxidative stress also depends on AP-1, mediated by the MAPK signaling system of JNK. We speculate that the upregulation of these three enzymes in the intact porcine lens epithelial cell layer may also use this mechanism. 

Sun et al. ³⁵ exposed human epidermoid carcinoma A431 cells to H₂O₂ (0.2 mM) for 4 hours and found an approximate fivefold increase in TR-1 but no effect on Trx-1 expression. In our present study, we observed a ∼2-fold increase in gene expression for all three enzymes (Figs. 4B 5B 6B) . The magnitude of Trx expression is in agreement with our previous results using cultured HLE B3 cells. ¹¹ This low level of increase may have physiological implications, as Berggren et al. ¹⁵ have shown that nearly 100-fold increase of Trx expression was found in human primary colorectal carcinomas, and a 10- to 23-fold increase was found in several human cancer cell lines. These findings indicate that abnormally high levels of Trx may not be compatible with the normal physiological conditions of the cells. Regulatory mechanisms such as binding of Trx by Trx-binding protein (TBP)-2 has been found to regulate Trx negatively in cells ⁴⁴ ; however, no regulatory mechanism has been reported for TTase or TR to date. 

Increased activity and expression of TTase under sustained oxidative stress conditions are essential to protect the cells, as this oxidant-resistant dethiolase enzyme can repair the oxidation-damaged key enzymes and proteins, such as the adenosine triphosphate (ATP)-generating G3PD and H₂O₂ detoxifying enzyme, glutathione peroxidase, as shown by Xing and Lou. ⁴⁵ In addition, increased TTase activity can enhance ascorbate recycling capability to diminish the accumulation of the toxic dehydroascorbate in the cells. ⁹ Elevated Trx and TR under oxidative stress would be expected to help lens cells to recover from oxidative damage, as Trx is capable of regenerating oxidatively damaged proteins/enzymes by disrupting the disulfide bonds ⁸ and removing toxic H₂O₂ through donation of electrons for peroxiredoxins to hydrolyze H₂O₂. ⁴⁶ Methionine sulfoxide reductase is another Trx-dependent protein repair system in the lens ⁴⁷ that is capable of repairing oxidatively damaged methionine residues in proteins. This repair system may also be upregulated under our experimental conditions and together with the Trx/TR system may repair oxidatively damaged proteins/enzymes. However, under persistent oxidative stress conditions, enzyme repair systems may be severely inactivated. Therefore, we anticipate that severe damage to the lens epithelium under our experimental conditions would result in cataract formation, as was observed in this study. 

In summary, in our study, TTase, Trx, and TR were very resistant to oxidative stress and were transiently upregulated under oxidative stress conditions. As far as we know, this is the first time that the oxidative stress–induced activation of these two oxidation repair systems in the lens has been demonstrated. The mechanism of the oxidation-associated gene regulation for these enzymes should be an interesting subject for further study. 

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2003.

 Supported by Grant R01EY10595 from the National Eye Institute (MFL).

 Submitted for publication February 22, 2005; revised May 29 and June 15, 2005; accepted August 12, 2005.

 Disclosure: S. Moon, None; M.R. Fernando, None; M.F. Lou, 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.

 

The effect of various concentrations of H₂O₂ on epithelial GSH level in cultured porcine lenses. Porcine lenses were incubated and harvested at indicated times. Three epithelial layers were pooled, homogenized, and used for GSH quantification. Lenses in group onewere incubated in TC-199 medium alone, lenses in group two were incubated in medium containing 0.2 mM H₂O₂, and lenses in group three were incubated in medium containing 0.5 mM H₂O₂. Data are the average of five determinations. Error bars, SEM.

Figure 2.

 

Morphology of porcine lenses cultured in the presence and absence of H₂O₂. Porcine lenses were preincubated in TC-199 medium for 2 hours before being transferred to 12-well plates and incubated individually in 4 mL fresh TC-199 medium. The lenses were divided into three groups with three lenses in each. Group one or the control group was incubated in medium without oxidative stress; removed at 0, 1, 6, 12 and 24 hours; and photographed and observed for morphologic changes. The other two groups were incubated in medium containing 0.2 and 0.5 mM H₂O₂. Lenses in these two groups were treated the same way as those in the control group. The images of the lenses shown are representative of each group.

Figure 3.

 

The distribution of TTase activity in various regions of the lens. Three fresh porcine lenses were dissected into the epithelial layer, outer cortex, inner cortex, and nuclear regions and pooled. These tissue fractions were homogenized, total soluble fractions were obtained, and TTase activity was determined. Data are the average of four determinations. Error bars, SEM.

Figure 4.

 

The effect of H₂O₂ on TTase activity and expression in cultured porcine lenses. Fresh porcine lenses were divided into three groups with three lenses per group. Group one (control) was incubated in the absence of oxidant, groups two and three were was incubated in medium containing 0.2 mM H₂O₂ and 0.5 mM H₂O₂, respectively. Lenses were taken at indicated times, epithelial layers were removed, and the total soluble fraction prepared. (A) The lysate of the pooled three lens epithelial layers of each group was obtained at 0, 1, 2, 4, 6, 9, 12, 18, and 24 hours and analyzed for TTase activity. Data are the average of six determinations. Error bars, SEM. (B) Total soluble fractions (40 μg of protein) from the lysates in (A) were subjected to immunoblot analysis with antibody specific to TTase. GAPDH was used as the internal control.

Figure 5.

 

The effect of H₂O₂ on Trx activity and expression in cultured porcine lenses. Fresh porcine lenses were obtained and incubated in the absence (control) or presence of 0.2 or 0.5 mM H₂O₂ for the indicated times as described in Figure 4 . Lenses were taken at the indicated times, epithelial layers were removed, and the total soluble fraction prepared. (A) The lysates of lens epithelial layers of all three groups were obtained at the indicated times and analyzed for Trx activity. Data are the average of six determinations. Error bars, SEM. (B) Total soluble fractions (40 μg of protein) of the lysates in (A) from various time points were subjected to immunoblot analysis with antibody specific to Trx. GAPDH was used as the internal control.

Figure 6.

 

The effect of H₂O₂ on TR activity and expression in cultured porcine lenses. Fresh porcine lenses were obtained and incubated in the absence (control) or presence of 0.2 or 0.5 mM H₂O₂ for the indicated times described in Figure 4 . Lenses were taken at the indicated times, the epithelial layers were removed, and the total soluble fraction prepared. (A) The lysates of lens epithelial layers of all three groups were obtained at the indicated times and analyzed for TR activity. Data are the average of six determinations. Error bars, SEM. (B) Total soluble fractions (40 μg of protein) of the lysates from various time points were subjected to immunoblot analysis with antibody specific to TR. GAPDH was used as an internal control.

Figure 7.

 

The effect of H₂O₂ on TTase, Trx, and TR mRNA upregulation in cultured porcine lens epithelial layers. Fresh porcine lenses were obtained and incubated in the presence or absence of 0.5 mM H₂O₂ for the indicated times (n = 3 lenses per group). Lenses were removed at the indicated times and thoroughly rinsed and epithelial layers removed. Total RNA was extracted from the three pooled epithelial layers (in each group) and reverse transcribed. Primers were designed and synthesized to detect TTase, Trx, TR, and β-actin. Equal amounts of synthesized cDNA were amplified by PCR with synthesized primers. PCR products were analyzed by agarose gel (1%) electrophoresis. β-Actin was amplified as the internal control.