SOD2 contributes to anti-oxidative capacity in rabbit corneal endothelial cells

Cell culture

Rabbit corneal endothelial (RCE) cells were isolated from fresh peeled corneal endothelium of young (8 weeks) New Zealand white rabbit eyes (Pel-Frez Biologicals, Roger, AR). Peeled endothelium was incubated in disaggregating solution (300 Units type I collagenase, 100 Units hyalronidase, and 1% antibiotic/antimycotic) in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) for about 3 h at 37 °C. CE cells were collected by centrifugation at 450× g for 5 min. Cells from four corneas were suspended in a 35 mm Petri dish with 2 ml DMEM supplemented with 10% bovine calf serum (Thermo Scientific, Logan, UT) and 1% antibiotic-antimycotic. Cells were incubated at 37 °C with 5% CO₂ and passaged 3 days later when they reached 90% confluence.

siSOD2 design and transfection

Two sense and antisense oligonucleotides corresponding to Oryctolagus cuniculus (rabbit) manganese superoxide dismutase (SOD2) mRNA (Genebank L28808.1, 5′-AAA CGT CAG ACC TGA TTA TCT-3′ (525–545) and 5′-AAT GTA ACT GAA AGA TAC ATG-3′ (577–597) were designed by an Ambion siRNA targeting design tool (Ambion, Austin, TX) and blasted by NCBI_BLAST. siSOD2 was synthesized using the Silencer siRNA construction kit (Ambion, Austin, TX). An siControl nontargeting siRNA (no known mammalian homology) was purchased from Invitrogen (Carlsbad, CA). Fifty thousand cells/well were seeded in 12-well plates and transfected with 10 nM siSOD2 or siControl by using Lipofectamine^TM 2000 (Invitrogen). Transfected cells were incubated with 1 ml of Opti-MEM (Invitrogen) containing siRNA for 4 h, followed by addition of 1 ml of standard DMEM with 2% serum. Transfected cells were harvested 68 h post transfection for detecting knockdown efficiency by western blot, enzyme activity assay, and ROS production assay. Based on SOD2 knockdown results, a sequence targeting 5′-AAA CGT CAG ACC TGA TTA TCT-3′ (525–545) was selected for the remainder of the experiments and used for shSOD2 design.

shSOD2 pseudo-lentivirus production and transduction

Lentiviral vectors were produced to test the relative knockdown efficacy with an eye to using them in future in vivo applications. shSOD2 was designed following the manufacturer’s (System Biosciences, SBI, Mountain View, CA) instructions with primers: 5′-GAT CCA AAC GTC AGA CCT GAT TAT CTC TTC CTG TCA GAA GAT AAT CAG GTC TGA CGT TTT TTT TG-3′ and 5'-AAT TCA AAA AAA ACG TCA GAC CTG ATT ATC TTC TGA CAG GAA GAG ATA ATC AGG TCT GAC GTT TG-3'. An shRNA targeting wild type firefly Luciferase gene was designed as a scrambled control (5′-GAT CCG TGC GTT GTT AGT ACT AAT CCT ATT TGT GAA GCA GAT GAA ATA GGG TTG GTA CTA GCA ACG CAC TTT TTG-3′ and 5′-AAT TCA AAA AGT GCG TTG CTA GTA CCA ACC CTA TTT CAT CTG CTT CAC AAA TAG GAT TAG TAC TAA CAA CGC ACG-3′). shSOD2 and shLuciferase template oligonucleotides were synthesized (Integrated DNA Technologies, IDT, Coralville, IA) and cloned into feline immunodeficiency virus (FIV) based pSIF-H1-Puro shRNA vectors (SBI). As described previously [26], lentiviral constructs pSIF-H1-Puro-shSOD2 and pSIF-H1-Puro-shLuciferase were individually packaged into lentiviral transducing particles which were pseudotyped with vesicular stomatitis virus G (VSV-G) protein (pPACK ^TM lentivector packaging kit, SBI). Briefly, lentivirus vector was combined with the mixture of pFIV-34N and pVSV-G plasmids, and then transfected into 293TN HEK cell by using Lipofectamine ^TM Reagent (Invitrogen). Pseudovirus containing medium was collected 48 and 72 h post transfection followed by purifying pseudo-lentiviral particles with PEG-it virus precipitation solution (SBI) overnight at 4 °C. The viral particle preparation was centrifuged at 1,500× g for 30 min at 4 °C. Residual PEG-it solution was removed by another centrifugation at 1,500× g for 5 min. Viral particles were washed and then resuspended in cold, sterile phosphate buffered saline (PBS). Virus stocks were aliquoted and stored at −80 °C. To determine virus titer, serially dilutions of stocks were inoculated to 293TN HEK cells. Seventy-two hours post viral transduction, cells were collected for a real time PCR-based titration (UltraRapid Lentiviral Titer Kit; SBI) based on the amplification of a small fragment from the lentivector-specific WPRE (Woodchuck hepatits virus post transcriptional Regulation Element) that is integrated into the genome of transduced cells.

Fifty thousand RCE cells/well were seed in 12-well plates overnight and transducted with shRNA virus at an m.o.i. of 20 in the presence of 7 mg/ml of Polybrene (Hexadimethrine Bromide; Sigma, St. Louis, MO). Cells were collected at 5 days post transduction for western blot analysis, enzyme activity assay, ROS production assay, mitochondrial membrane potential and cell apoptosis assay.

RT–PCR

Total RNA was extracted from freshly dissected corneal endothelium or cultured RCE using an RNeasy Micro Kit (Qiagen, Valencia, CA). RT–PCR was performed using SuperScript™ One-Step RT–PCR with Platinum® Taq (Invitrogen) with the primers of 5′-CTC CCC GAC CTG CCC TAC GAC-3' and 5'-TGC AGG TAG TAA GCG TG-3'. RT–PCR reactions comprised 30 min reverse transcription at 50 °C for 30 min, 2 min of initial denaturation at 94 °C. 40 cycles of denature (94 °C for 15 s), annealing (58 °C for 30 s) and extension (72 ^o C for 1 min), followed by a 5-min final extension at 72 °C.

Western blot

SOD2 protein expression level was determined by western blot analysis as previously described [26], β-actin in the same sample was detected as an internal control. RCE cells were washed with cold PBS and then extracted with RIPA lysis buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) with complete protease inhibitor cocktails (Sigma, St Louis, MO). The preparation was briefly sonicated (Branson Sonifier 250, Dunbury, CT) on ice and centrifuged at 10,000× g for 5 min. An aliquot of the supernatant was taken for protein concentration measurement using the BCA protein assay (Bio-Rad, Hercules, CA). Samples were separated on SDS–PAGE gels and electroblotted to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Based on the prestained protein markers (Precision Plus protein standards; Bio-RAD), blots were horizontally cut into two parts just above the 37 kDa prestained marker. The lower part with protein sizes less than 37 kDa was probed with SOD2 polyclonal antibodies (Millipore/Upstate, Billerica, MA) at 1:2,000. The upper part was probed with β-actin monoclonal antibody (Sigma) at a dilution of 1:10,000. Enhanced chemiluminescence (ECL, Thermo Scientific/Pierce, Rockford, IL) was used for detection. Films were scanned to produce digital images that were then subjected to densitometry analysis (UN-SCAN-IT gel 6.1; Silk Scientific Corporation, Orem, UT). SOD2 protein levels in test samples were normalized with β-actin (ratio of SOD2 to β-actin). The mitochondrial/cytosolic fractions from RCE cells were separated using a fractionation kit (BioVision, Mountain View, CA). The same amount of protein from each fraction was loaded for western blot analysis. The molecular weight for SOD2 and β-actin is 24 kDa and 43 kDa, respectively.

Enzyme activity assay

RCE cells were collected in ice-cold PBS (pH 7.2) that contained a protease inhibitor cocktail (Sigma) by centrifugation for 10 min at 250× g at 4 °C. Cells were briefly sonicated on ice and centrifuged for 5 min at 12,000× g at 4 °C. The supernatant was used immediately for activity assay. Protein concentration was measured by BCA protein assay kit. SOD2 activity was detected by using a superoxide dismutase assay kit (Cell Technology, Mountain view, CA). This assay utilizes a highly water-soluble tetrazolium salt, WST-1 (2-(4-Iodophenyl) - 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon reduction with a superoxide anion. The rate of the reduction with superoxide anion is linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD. Reduction in the appearance of WST-1-formazan is a measure of SOD activity present in the experimental samples. WST-1 reduction in siRNA- or shRNA- treated cells was compared to that in siControl- or scrambled shRNA-treated cells, this ratio was calculated as the relative SOD2 activity. Each reaction contained 500 ng protein sample, 600 μl WST-1, 30 μl XO, 1 mM KCN, add XO buffer to a final volume of 800 μl. The reaction without cell lysate was used as a blank control. Each reaction was performed in 40 min at 37 °C. The initial rate of reduction in the appearance of WST-1-formazan was followed by measuring the A₄₄₀ using ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). One millimolar KCN was used to block the activity of SOD1 and SOD3.

ROS production assay

Mitochondrial ROS in siSOD2 and siControl transfected cells was compared by monitoring MitoSOX™ (Invitrogen/Molecular Probes, Eugene, OR) fluorescence intensity. Ten thousand cells were seeded on a coverslip in a six-well plate and grown overnight. Cells were loaded with 1 µM MitoSOX™ and incubated at 37 °C for 10 min followed by three washes with warm PBS. MitoSOX™ intensity was recorded with a standard epifluorescence microscope (E600; Nikon, Tokyo, Japan) equipped with a cooled charge-coupled device camera (Princeton Instruments, Princeton, NY).

ROS production in shRNA lentiviral treated cells was measured by flow cytometry. Five days post transduction, cells were washed with Hanks’ Balanced Salt Solution (HBSS, Invitrogen) and then loaded with 5 µM 5-(and-6) - carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA; Invitrogen/Molecular Probes) and incubated at 37 °C for 30 min. Cells were detached by 0.25% trypsin and then spun at 500× g for 5 min followed by suspending in FACS buffer for measurement. The percentage of carboxy-H₂DCF stained cells was analyzed by a FACS-Calibur flow cytometer (BD biosciences, San Jose, CA) using CellQuestPro (BD Biosciences) software. A positive control for ROS production was produced by exposing CE to a UV germicidal lamp for 2 min in a laminar biosafety carbinet (Labconco, Janesville, WI). A representative line histogram is graphed by flowJo (version7.6; BD Biosciences) to show the DCF fluorescence intensity shift.

Mitochondrial membrane potential (MMP) and cell apoptosis assay

MMP was determined with 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazole carbocyanide iodine (JC-1; Invitrogen/Molecular Probes) by using a BMG FLUOstar microplate reader (BMG, Alexandria, VA). Fifty thousand cells per well were grown overnight in 12-well plates in DMEM media. Cells were loaded with 100 nM JC-1 and incubated at 37 °C for 15 min followed by wash with warm HBSS solution. JC-1 was excited at 488 nm, and emission was determined at 529 and 585 nm.

Cell apoptosis was measured by using Annexin V-FITC (BioVision). Annexin V-FITC (5 μl) was added to each well and incubated at room temperature for 5 min in the dark. Fluorescence was measured by microplate reader with ex/em of 488/530. Cells were then collected for BCA protein assay (Thermo scientific/Pierce) to normalize fluorescence per microgram cell protein.

Statistical analysis

Results are expressed as means±SEM from at least three replicates. Data was analyzed by a two-tailed Student’s t-test and one-way ANOVA analysis. A p value <0.05 was regarded as a significant difference.

SOD2 expression and knockdown

RT–PCR (Figure 1A) and western blot analysis (Figure 1B) confirmed that SOD2 is expressed in both fresh and cultured rabbit corneal endothelial cells. Moreover, SOD2 expression is maintained from passage one to passage two in cultured cells. Figure 1C shows that SOD2 is predominately associated with the mitochondrial fraction.

Two siSOD2 sequences were tested to knockdown rabbit SOD2 mRNA (Genebank ID L28808.1): 5′-AAA CGT CAG ACC TGA TTA TCT-3′ (525–545) and 5′-AAT GTA ACT GAA AGA TAC ATG-3′ (577–597). Western blot analysis showed that the sequence 5′-AAA CGT CAG ACC TGA TTA TCT-3′ could efficiently knockdown SOD2 expression with a reduction of 80% in corneal endothelial cells (Figure 2A). This sequence was used for the remaining experiments and shSOD2 design. Primary cultured RCE cells were transfected with shSOD2 lentiviral pseduoparticles with an m.o.i of 20. Five days after viral transduction, cells were collected for detecting SOD2 expression level. Western blot analysis demonstrated that shSOD2 lentiviral transduction is able to reduce SOD2 expression by ~90% in cultured cells (Figure 2B).

SOD2 enzyme activity

To test the effect of SOD2 knockdown on enzyme activity, SOD2 enzyme activity was measured by monitoring the change of substrate WST-1 formazan absorbance. Figure 3 shows that SOD2 activity was not significantly different between untreated cells and scrambled RNA treated RCE. However, SOD2 activity was reduced by 81% and 71% in siSOD2 and shSOD2-lentiviral treated cells, respectively.

ROS production

Next we asked whether a reduced SOD2 activity affects ROS levels. Mitochondrial ROS was qualitatively detected by MitoSOX™ staining and total ROS by DCF flow cytometry approaches. Figure 4A shows that MitotSox™ fluorescence intensity in siSOD2 treated RCE cells was dramatically increased relative to that in scrambled sequence treated cells. Figure 4B is a representative histogram and shows that total ROS, as measured by DCF fluorescence also increased following SOD2 knockdown. The DCF fluorescence intensity shift to the right in shSOD2 virus treated cells indicates an increased ROS production. Figure 4C summarizes DCF flow cytometry analysis. Significant DCF fluorescence was present in 0.4%±0.1% of untreated cells and scrambled-sequence virus treated cells. However, DCF fluorescence positive cells significantly increased to 9%±6% in shSOD2 virus treated cells (p<0.05), a 22.5 fold increase.

Next, we examined the role of SOD2 in clearing ROS following a burst of ROS production induced by 2 min exposure to a UV germicidal lamp. With 2 min UV irradiation, untreated cells and scrambled sequence treated cells yielded similar levels of DCF positive fluorescence (~15%, Figure 4C), whereas shSOD2 treated cells showed dramatically increased DCF fluorescence (29%±5%, p<0.05). Flow cytometry results indicated that SOD2 knockdown contributed to ROS generation in RCE cells, while increasing sensitivity to oxidative stress (UV).

Mitochondrial membrane potential (MMP) and cell apoptosis

The effect of SOD2 knockdown on MMP was detected with the cationic dye JC-1. In the undamaged mitochondria with normal membrane potential, JC-1 aggregates produce a red fluorescence, whereas in cells with depolarized MMP, the dye remains as monomers producing a diffuse green fluorescence. The ratio of red/green fluorescence is a measure of MMP, with a decrease in the ratio indicating depolarization. Figure 5A shows that five days post viral transduction MMP in scrambled virus treated cells was about 15%±8% lower than untreated cells. However in shSOD2 viral treated cells MMP was depolarized by 66%±3% (p<0.05). As a positive control MMP was depolarized by treatment with CCCP, a protonophore. This produced 88%±2% decrease in JC-1 fluorescence ratio. These results indicate that the decreased level of SOD2 expression indirectly induces a depolarization of MMP.

Cellular redox state plays a critical role in programmed cell death. An increased ROS level frequently represents a triggering event upstream of the mitochondrial membrane depolarization, cytochrome c release, caspase activation, and nuclear fragmentation [5,27]. Our results showed that knockdown of SOD2 elevated ROS production and induced mitochondrial membrane potential depolarization suggesting that SOD2 knockdown would increase cell apoptosis. shRNA virus treated cells were collected at 5 days post virus transduction for detecting early apoptosis using Annexin V-FITC staining. Figure 5B shows that this assay detected a significantly higher level (1.5 fold, p<0.05) of Annexin V-FITC fluorescence in shSOD2 virus treated samples compared with scrambled virus and untreated cells, indicating that a reduced SOD2 level contributes to early apoptosis in RCE cells.