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Biochemistry and Molecular Biology  |   July 2013
Regulation of iNOS Expression by NF-κB in Human Lens Epithelial Cells Treated With High Levels of Glucose
Author Affiliations & Notes
  • Jinhai Jia
    Clinic of Hebei Medical University, Hebei Medical University, Shijiazhuang, China
  • Yuan Liu
    Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, Shijiazhuang, China
  • Xiaolin Zhang
    Department of Epidemiology and Statistics, Hebei Medical University, Shijiazhuang, China
  • Xinke Liu
    Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, Shijiazhuang, China
  • Jinsheng Qi
    Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, Shijiazhuang, China
  • Correspondence: Jinsheng Qi, Department of Biochemistry, Hebei Key Laboratory of Medical Biotechnology, Hebei Medical University, No. 361 East Zhongshan Road, Shijia-zhuang 050017, Hebei, China; qijinsheng777@163.com
Investigative Ophthalmology & Visual Science July 2013, Vol.54, 5070-5077. doi:10.1167/iovs.13-11796
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      Jinhai Jia, Yuan Liu, Xiaolin Zhang, Xinke Liu, Jinsheng Qi; Regulation of iNOS Expression by NF-κB in Human Lens Epithelial Cells Treated With High Levels of Glucose. Invest. Ophthalmol. Vis. Sci. 2013;54(7):5070-5077. doi: 10.1167/iovs.13-11796.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To explore the regulation of inducible nitric oxide synthase (iNOS) expression by nuclear factor kappa B (NF-κB) in human lens epithelial cells (LECs) treated with high levels of glucose, and to elucidate the impact of this in the pathogenesis of cataracts associated with diabetes.

Methods.: LECs (SRA01/04) were cultured in vitro. NF-κB nuclear translocation and iNOS expression were measured at different glucose concentrations and at various time points, and the optimal concentration for detecting changes in the patterns of NF-κB nuclear translocation and iNOS expression was chosen. As a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) was used to assess the effect of inhibiting NF-κB. Western blotting and inverted fluorescence microscopy were used to monitor the nuclear translocation of NF-κB. PCR and Western blotting were used to measure iNOS expression. Using the University of California, Santa Cruz database and the TFSEARCH program, we searched the DNA sequence upstream of iNOS for the core binding sequence for NF-κB. Chromatin immunoprecipitation (ChIP) was used to measure the binding of NF-κB.

Results.: The nuclear translocation of NF-κB was measured upon glucose treatment, and the concentration of NF-κB in the nucleus was found to peak at 25 to 30 minutes of treatment with 25 mM glucose. iNOS mRNA and protein levels also increased significantly in a time- and concentration-dependent manner and iNOS mRNA and protein reached their peak values after 8 hours of treatment with 25 mM glucose. The binding of NF-κB to the promoter of the iNOS gene was enhanced in the 25 mM glucose group compared with the 5.5 mM glucose group or the 25 mM glucose + 100 μL PDTC group, and this difference was statistically significant (P < 0.05).

Conclusions.: NF-κB regulates iNOS expression in a time- and concentration-dependent manner. Under high glucose conditions, NF-κB is activated and rapidly translocates to the nucleus, leading to increased binding to the iNOS promoter and a consequent increase in iNOS expression. The findings of this study provide important experimental evidence that clarifies the pathogenesis of cataracts associated with diabetes and contributes to the search for therapeutic targets of these cataracts.

Introduction
Diabetic cataract conditions are among the most common complications of diabetes and are a major cause of visual impairment in diabetic patients. 1 The incidence and progression of cataracts in diabetic patients tend to increase with the severity of diabetes mellitus. 2 Cataract surgery remains the only cure for diabetic cataracts, but the postoperative complications and the surgical costs make it an unsatisfactory choice; therefore, a better understanding of the mechanisms underlying diabetic cataracts is a high priority. 37  
Many factors, such as the polyol pathway, nonenzymatic glycation and glycoxidation, oxidative–nitrosative stress, and inhibition of lens gap junctions contribute to diabetic cataract. 815 Damage to the lens epithelial cells (LECs) is considered to be the key factor in diabetic cataract pathogenesis. 16 The human lens is a nonvascularized and noninnervated organ, and it contains only a single layer of epithelial cells at its anterior surface. This epithelium is thought to protect underlying fibers from injury, 17 and it maintains the transparency of the lens. 10,18  
Oxidative stress is the final common pathway for all the chronic complications of diabetes, and it is the decisive cause of the occurrence and development of diabetic cataract, an observation that we confirmed in our previous study. 1923 Inducible nitric oxide synthase (iNOS) is a key enzyme for oxidative stress, and once induced, iNOS can generate excessive nitric oxide (NO) and superoxide anions (O2 ), 20,24 leading to extensive oxidative damage. 2529  
Nuclear factor-κB (NF-κB), which is a key factor in the oxidative stress reaction, is located in the cytoplasm (in the form of p65/p50 heterodimers) in its nonactivated state. When stimulated, NF-κB is activated and translocates into the nucleus. It then binds to specific sites in DNA to activate gene transcription. 30,31  
Increased expression of iNOS has been detected in many eye diseases. 20,3237 Previous research has revealed that both NF-κB and iNOS expression are increased in the LECs of Zucker fatty rats. 38 However, it remains unclear whether NF-κB regulates iNOS expression in human LECs at high glucose levels. 
Therefore, in this study, the LEC line SRA01/04 was used to explore the regulation of iNOS under high glucose conditions. Western blotting and inverted fluorescence microscopy were used to detect nuclear translocation of NF-κB. Western blotting and PCR were used to measure the level of iNOS expression. Additionally, chromatin immunoprecipitation (ChIP) was used to measure the binding of NF-κB to the iNOS promoter. Based on this study, we demonstrated that NF-κB is activated under high glucose conditions and binds to the iNOS promoter, thereby elevating the expression of iNOS. 
Materials and Methods
Materials
Trizol reagent box (TianGen Corporation, Beijing, China); RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas, Ottawa, Canada); ChIP Assay Kit (Millipore, Boston, MA); mouse monoclonal anti-human iNOS antibody; mouse monoclonal anti-human β-actin antibody (Santa Cruz Corporation, Dallas, TX); mouse monoclonal anti-human NF-κB p65 antibody (Thermo Corporation, Waltham, MA); rabbit monoclonal anti-human Histone H2A.X antibody (Bioworld Corporation, Atlanta, GA); goat blood serum; rabbit monoclonal anti-human antibody; tetramethyl rhodamine isothiocynate (TRITC) labeled anti-mouse antibody (Beijing Zhongshan Biological Technology Limited Company, Beijing, China); fluorescein conjugated anti-mouse IgG (H&L) (goat) antibody; fluorescein conjugated Anti-rabbit IgG (H&L) (goat) antibody (Rockland Corporation, Gilbertsville, PA); 4′, 6-diamidino-2-phenylindole (DAPI; Roche Corporation, Basel, Switzerland); benzyl sulfonic acid fluoride (Amresco Corporation, Solon, OH); newborn cow blood serum (PAA Corporation, Houston, TX); non-essential amino acids (PAA Corporation); low glucose DMEM (Gibco Corporation, Carlsbad, CA); trypsin (Gibco Corporation); dithiothreitol (Merck Corporation, Whitehouse Station, NJ); PDTC (Sigma Corporation, Santa Clara, CA); and ethylene glycol diethylether diamidogen four ethanoic acid (Sigma Corporation). CO2 incubator (Heraeus Company, Hanau, Germany); Clean Benches (Shanghai BoXun Industrial Company, Shanghai, China); ultra-low temperature refrigerator (Sanyo, Osaka, Japan); inverted fluorescence microscope (Chongqing Optical Instrument Factory, Chongqing, China); 3K30 low-temperature centrifuge (Sigma Corporation); gel imaging Analysis System (Nanjing Jieda Company, Nanjing, China); FS-150 ultrasonic cell crusher (Shanghai Ultrasonic Instrument Company, Shanghai, China); PH211 pH meter (Hanna, Villafranca Padovana, Italy); Odyssey near-infrared two-color laser imaging system (Gene Company, Hong Kong, China); and NanoDrop ND-1000 nucleic acid protein analyzer (NanoDrop company, Wilmington, DE). 
Cell Culture
In this study, the human lens epithelial cell line SRA01/04 was obtained from the Cancer Institute & Hospital, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in low glucose DMEM culture medium with 10% newborn calf serum in an incubator with 5% CO2 at 37°C. When the cells were approximately 80% confluent, they were passaged. 
Description of Groups
To detect the nuclear translocation of NF-κB p65, the SRA01/04 cells were treated with glucose at various concentrations: 5.5, 10, 15, 20, 25, and 30 mM. The glucose concentration eliciting the largest response was chosen, and cells were further divided into the following nine time groups: 0, 5, 10, 15, 20, 25, 30, 35, and 40 minutes. Cells were also treated with the optimal glucose concentration + PDTC (100 μM) for the same nine periods of time. 
To measure the expression of iNOS, the SRA01/04 cells were divided into the following four groups according to glucose concentration: 5.5, 15, 25, and 30 mM groups. The optimal glucose concentration was chosen and the cells divided into the following five time groups: 0, 1, 2, 4, and 8 hours. Cells were also treated with the optimal glucose concentration + PDTC (100 μM) for the same five periods of time. 
To detect NF-κB p65 binding to the iNOS promoter, the SRA01/04 cells were treated with 5.5 mM glucose, 25 mM glucose, and 25 mM glucose + 100 μM PDTC, respectively. ChIP technology was used to assay the activity of NF-κB p65 binding to the iNOS promoter. The groups were defined as follows: input group, NF-κB p65 antibody group (experimental group), Histone H2A.X antibody group (positive antibody control group), IgG antibody group (negative antibody control group), and no antibody group (negative control). The input group was used as an internal reference, and the process used for the input group was identical to that used for the experimental groups (except for the immunoprecipitation step). 
Primer Design
Primers for iNOS, β-actin, and the NF-κB binding sites in the iNOS promoter were designed using the Primer premier 5.0 software (Premier Biosoft, Palo Alto, CA). The annealing temperatures and primer oligonucleotide sequence specificities are shown in the Table
Table
 
Primer Sequences Employed for PCR
Table
 
Primer Sequences Employed for PCR
Gene Name Primer Sequences Product Size Annealing Temperature
iNOS Upstream 5′-CCTCGGCTCCAGCATGTACCCTCGG-3′ 135 bp 60°C
Downstream 5′-CGGAAGGCGTCCTCCTGCCCACTGA-3′
iNOS promoter Upstream 5′-CCACCCTTGGATACCGCAT-3′ 430 bp 53°C
Downstream 5′-CTCCCACTCCTACCCATTTCT-3′
β-actin Upstream 5′-TCGGGGCATCGGAACCGCTCA-3′ 404 bp 60°C
Downstream 5′-GAGACCTTCAAGACCCCAGCC-3′
Observing NF-κB p65 Nuclear Translocation With Inverted Fluorescence Microscopy
The cells were seeded on 6-well plates, and a sterile coverslip was placed at the bottom of each well. When confluent, cells were treated with various concentrations of glucose (with or without PDTC). Next, the cells were fixed in 4% paraformaldehyde for 15 minutes, washed 3 times with PBS, incubated at room temperature in 1% TritonX-100 for 30 minutes, and then washed 3 times with PBS. Afterwards, the plates were incubated in 10% goat serum at room temperature for 1 hour. Next, 100 μL of the NF-κB p65 antibody (1:200 dilution) was added to each well. Then, the plates were incubated at 37°C for 2 hours, 100 μL of fluorescent secondary antibody (1:100 dilution) was added to each well and the plates were incubated at 37°C for 30 minutes. The cells were then washed with PBS 3 times before adding 100 μL of 0.5 μg/mL DAPI to each well. After the plates were incubated at room temperature for 15 minutes, they were washed 3 times with PBS. Then, the plates were rinsed with 50% glycerol phosphate buffer, and the stained specimens were examined by inverted fluorescence microscopy. The experiment was validated using control groups lacking either the primary antibody or the secondary antibody. 
Detection of iNOS and NF-κB p65 Protein by Western Blotting
The protocol for cell protein extraction was performed as follows: the medium was discarded, the cells were rinsed with ice cold PBS 3 times and then the cells were lysed with 300 μL lysis buffer (50 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4•12H2O, 1.5 mM KH2PO4, 0.35 mM SDS). The sample was centrifuged at 14800g for 5 minutes, and the cellular proteins were harvested from the supernatant. The following steps for nuclear protein extraction were performed: the cells were treated with lysis buffer A (10 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DL-dithiothreitol [DTT], 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride [PMSF]), vortex-mixed for 15 seconds, and incubated on ice for 15 minutes; NP-40 was then added to a final concentration of 0.6%. After shaking the sample for 3 seconds and centrifuging at 14800g for 5 minutes at 4°C, the centrifugation sediment was resuspended in lysis buffer B (20 mM HEPES-KOH pH 7.9, 400 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM Na3VO4, 1 mM PMSF), and the sample was mixed on a vortex for 15 seconds. After 20 minutes on ice, the sample was centrifuged at 14800g for 20 minutes at 4°C, and the nuclear proteins were harvested from the supernatant. 
The nuclear proteins were examined for NF-κB p65, and the cellular proteins were examined for iNOS. Equal amounts of protein (50 μg) per sample were loaded onto 10% SDS-PAGE gels for electrophoresis and transferred onto a PVDF membrane under a voltage of 80 V for 5 hours. Wet transferred PVDF membranes were blocked in 5% skim milk at 37°C for 2 hours. NF-κB p65, iNOS, β-actin, and H2A.X antibodies (diluted 1:4000, 1:200, 1:500, and 1:1000, respectively) were added dropwise to the PVDF membranes. The membranes were incubated at room temperature for 1 hour, then at 4°C overnight. The membranes were washed 3 times with 0.5% Tween 20 in PBS, incubated with fluorescent secondary antibody at room temperature for 1 hour, and then washed three times with 0.5% Tween-20 in PBS. An Odyssey near-infrared two-color laser imaging system (LI-COR Biosciences, Lincoln, NE) was used to visualize and measure the results. The relative content of iNOS was determined by normalizing iNOS to β-actin, and the relative content of NF-κB p65 was determined by normalizing NF-κB p65 to H2A.X. 
Detection of iNOS mRNA by RT-PCR
Total RNA was extracted from SRA01/04 cells using Trizol reagent, and the total RNA concentration was determined using a NanoDrop ND-1000 nucleic acid protein analyzer. In accordance with the RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas) instructions, 2 μg of the total RNA was reverse transcribed into cDNA. 
A 20 μL reaction mixture was prepared for each sample and contained the following components: 10 μL 2×Taq PCR Master Mix (TianGen Corporation), 1.0 μL each of the upstream and downstream primers, 2.0 μL cDNA and triple-distilled water up to the total volume. The PCR amplification was performed for 34 cycles, each cycle consisting of 2 minutes at 94°C, 30 seconds at 60°C, 30 seconds at 72°C, and concluded with a final extension cycle of 2 minutes at 72°C. The level of product formation was visualized by 1.5% agarose gel electrophoresis. The electrophoresis band optical density was measured using a Gel Analyzer (Nanjing Jieda Company) by photographing and grayscale scanning. The iNOS mRNA expression level for each specimen was expressed as the ratio of iNOS mRNA to β-actin mRNA. 
Detection of the NF-κB p65 Binding With the iNOS Promoter by ChIP
The cells were treated with 5.5 mM glucose, 25 mM glucose, or 25 mM glucose + 100 μL PDTC for 8 hours. Soon afterwards, the cells were formaldehyde-fixed, and the DNA was sheared to 20 to 1000 bp fragments by sonication (frequency of 100 KHz, cycled 3–4 times). In accordance with the operating instructions for the ChIP Assay Kit, the DNA was immunoprecipitated and each group was purified. PCR was performed using the DNA of each group as a template, with regions of the iNOS promoter sequence containing the p65 specific binding site as the primers. 
Statistical Analysis
All data are presented as the means ± SD. Statistical analysis was performed using an independent Student t-test for comparison of two groups or a one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons. P less than 0.05 was considered statistically significant. All computations were performed using the SPSS system (version 16.0; IBM, Armonk, NY). 
Results
In this study, the activity of NF-κB p65, the level of iNOS, and the binding activity of NF-κB to the iNOS promoter were detected in LECs that were treated with high levels of glucose. To ensure the validity of this study, PDTC, which is a specific NF-κB inhibitor, was used to assess the effect of inhibiting NF-κB. 
Nuclear Levels of p65 at Various Glucose Concentrations
The level of p65 in the SRA01/04 cell nuclei gradually increased with increasing glucose concentration: levels in the 10, 15, 20, and 25 mM groups were all higher than that in the 5.5 mM group (P < 0.05). However, the level of nuclear p65 declined in the 30 mM group compared with the 25 mM group (Fig. 1) (P < 0.05). 
Figure 1
 
Changes in the level of NF-κB p65 at various glucose concentrations ( ± s). H2A.X was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; #compared with the 25 mM group, P < 0.05; n = 8.
Figure 1
 
Changes in the level of NF-κB p65 at various glucose concentrations ( ± s). H2A.X was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; #compared with the 25 mM group, P < 0.05; n = 8.
Nuclear Levels of p65 at Various Times After Treatment With 25 mM Glucose
The level of p65 in the SRA01/04 cell nuclei gradually increased with increasing time: levels in 5, 10, 15, 20, and 25 minute groups were all higher than that in the 0 minute group (P < 0.05). However, the level of nuclear p65 declined in the 30 minute group compared with the 25 minute group (P < 0.05). Upon treatment with high glucose + PDTC, only small, nonsignificant changes were observed over time (P > 0.05). Levels of nuclear p65 were significantly lower in the groups treated with PDTC and high glucose than those treated with high glucose alone, at every time point except 0 minutes (Fig. 2) (P < 0.05). 
Figure 2
 
Levels of NF-κB p65 under high glucose (25 mM) and high glucose + PDTC conditions ( ± s). H2A.X was selected as an internal reference. *Compared with the 0 minute group, P < 0.05; △Compared with the 20 minute group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 2
 
Levels of NF-κB p65 under high glucose (25 mM) and high glucose + PDTC conditions ( ± s). H2A.X was selected as an internal reference. *Compared with the 0 minute group, P < 0.05; △Compared with the 20 minute group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
p65 Translocation
Immunofluorescence was used to analyze p65 nuclear translocation, and the results are shown in Figure 3A. Colored arrows mark the nucleus (blue), p65 (red), and the merged results (purple). The translocation of p65 in each group is shown in Figure 3B; it gradually increased starting at 10 minutes and reached peak levels at 25 minutes and then declined at 30 minutes. Compared with the 0 minute group, the differences were all statistically significant (P < 0.05). 
Figure 3
 
p65 nuclear translocation in high glucose conditions ( ± s). (A) Immunofluorescence imaging results: three white arrows indicate nuclei (blue), p65 (red), and merged results (purple), respectively. (B) Levels of p65 translocation after various incubation times. *Compared with the 0 minute group, P < 0.05; #compared with the 25 minute group, P < 0.05; n = 8.
Figure 3
 
p65 nuclear translocation in high glucose conditions ( ± s). (A) Immunofluorescence imaging results: three white arrows indicate nuclei (blue), p65 (red), and merged results (purple), respectively. (B) Levels of p65 translocation after various incubation times. *Compared with the 0 minute group, P < 0.05; #compared with the 25 minute group, P < 0.05; n = 8.
Changes in the Levels of iNOS at Various Glucose Concentrations
The levels of iNOS mRNA and protein were increased in the 15, 25, and 30 mM group. The differences were all statistically significant (P < 0.05) in comparison with the 5.5 mM group. The 30 mM group was slightly higher than the 25 mM group, but the difference was not statistically significant (Figs. 4, 5) (P < 0.05). 
Figure 4
 
Changes in the level of iNOS mRNA at various glucose concentrations ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 4
 
Changes in the level of iNOS mRNA at various glucose concentrations ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 5
 
Changes in the level of iNOS protein at various glucose concentrations ( ± s). β-actin was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 5
 
Changes in the level of iNOS protein at various glucose concentrations ( ± s). β-actin was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Changes in the Levels of iNOS at Various Times After Treatment With 25 mM Glucose
The time-dependent expression of iNOS mRNA and protein was examined and found to be in complete accordance with the pattern of NF-κB p65 nuclear transcription. Compared with the 0 hour group, the 1, 2, 4, and 8 hour groups had much higher iNOS levels, and the differences were all statistically significant (P < 0.05). In the high glucose + PDTC group, the level of iNOS mRNA and protein changed very little over time, and the differences were not statistically significant (P > 0.05). Levels of iNOS were significantly lower in the groups treated with PDTC and high glucose than those treated with high glucose alone, at every time point except 0 minutes (Figs. 6, 7) (P < 0.05). 
Figure 6
 
Levels of iNOS mRNA under high glucose and high glucose + PDTC conditions ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 6
 
Levels of iNOS mRNA under high glucose and high glucose + PDTC conditions ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 7
 
Levels of iNOS protein under high glucose and high glucose + PDTC conditions ( ± s). β-actin was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 7
 
Levels of iNOS protein under high glucose and high glucose + PDTC conditions ( ± s). β-actin was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Changes in NF-κB p65 Binding to the iNOS Promoter
As expected, no amplification products were detected in the control group or IgG group in either the low glucose or the high glucose condition, unlike the input and H2A.X-positive control groups. Furthermore, p65 amplification products were detectable only under high glucose, but not low glucose or high glucose + PDTC conditions. The level of p65 amplification was significantly different across the three groups (Fig. 8) (P < 0.05). 
Figure 8
 
Results of ChIP performed on cells in the low glucose, high glucose, and high glucose + PDTC groups ( ± s). *Compared with the low glucose and high glucose + PDTC groups, P < 0.05; n = 8.
Figure 8
 
Results of ChIP performed on cells in the low glucose, high glucose, and high glucose + PDTC groups ( ± s). *Compared with the low glucose and high glucose + PDTC groups, P < 0.05; n = 8.
Discussion
Worldwide, there are approximately 17 million people who lose their sight because of cataracts every year. The incidence of cataracts in diabetics is 2 to 4 times higher than the incidence in nondiabetics. 39,40 It is well known that lens epithelial cells are the core of the nutrient transportation, metabolism, and protein synthesis of the lens. Notably, LECs are responsible for lens regeneration and damage repair, and these cells play a key role in protecting the transparency and the internal environmental stability of the lens. Researchers have shown that, in diabetes mellitus, oxidative stress is the initiating factor that causes damage to lens epithelial cells and that this induces or accelerates the occurrence of diabetic cataract. 4144  
iNOS is regarded as a key enzyme associated with oxidative stress. Once induced, its activity can last up to 20 hours. The concentration of NO generated by iNOS is approximately 1000-fold higher than that of constitutive nitric oxide synthase. Excessive NO and O2 react rapidly to produce ONOO. Production of the ONOO species and its derivatives, whose oxidative capacity is 2000-fold stronger than H2O2, can lead to a severe oxidative stress reaction, causing extensive damage to lens epithelial cells. 27,28 Over-expression of iNOS had been detected in various eye diseases, 20,3237 as reported in our previous study. 45  
It is widely acknowledged that NF-κB is the major upstream regulator of iNOS. NF-κB consists of the following five family members: Rel-B, c-Rel, p50, p52, and, importantly, Rel-A (p65), which contains the transcription activation domain. 4648 Most NF-κB is present in the form of a p65/p50 dimer. Once activated, NF-κB can translocate into the nucleus rapidly and regulate specific genes. 31,49,50 In this study, the University of California, Santa Cruz Gene database and the TFSEARCH program were used to identify a core binding sequence (GGGAAGCCCC) for NF-κB lying upstream of the iNOS promoter region. However, it has not been reported how activation of NF-κB, iNOS expression, and NF-κB binding to the iNOS promoter are affected by high glucose levels. 
In this study, immunofluorescence and Western blotting showed that upon treatment with high glucose, NF-κB translocated into the nucleus (Fig. 3), and the level of p65 increased significantly (Figs. 1, 2), especially at 20 minutes after treatment. The PCR and Western blotting results showed that glucose treatment increased the expression of iNOS in a time- and concentration-dependent manner (Figs. 47). Furthermore, to ensure the credibility of the study, we used PDTC, a specific NF-κB inhibitor, to assess the effect of inhibiting NF-κB. 5153 The results of the Western blotting showed that the nuclear p65 and total iNOS levels decreased significantly in the high glucose + PDTC condition compared with the high glucose alone condition (Figs. 2, 6). 
Currently, the approaches used to study protein-DNA interactions include electrophoretic mobility shift assay, reporter gene analysis, DNA microarray, and ChIP. Of these, ChIP is the only one that is based on in vivo analysis and that can demonstrate the binding of a transcriptional regulator to its target gene(s) vividly and in real time. 5456 Therefore, ChIP was used to monitor the binding of NF-κB to the iNOS promoter in this study. To support our findings, we used the conservative H2A.X antibody as a positive control and an IgG antibody as a negative control. In addition, we used an input control to assess the effect of immunoprecipitation (i.e., the ChIP efficiency). Aside from a lack of immunoprecipitation, the experimental protocol for the input group was identical to the protocol for the experimental group. This study suggests that the key factor for improving ChIP efficiency is choosing a monoclonal antibody of high purity, specificity, and stability. The results for ChIP showed that the amplification products from the p65 group were almost undetectable in low glucose conditions and that their levels increased significantly in high glucose conditions and decreased significantly in the high glucose + PDTC condition (P < 0.05, Fig. 8), suggesting that high glucose treatment increases p65 binding to a specific site in the iNOS promoter. 
Overall, we have demonstrated that iNOS upregulation in SRA01/04 cells under high glucose conditions is closely related to the activation and nuclear translocation of NF-κB in the nucleus, which leads to the enhancement of iNOS promoter binding. The study provides evidence that elucidates the role in diabetic cataracts that NF-κB binding to the iNOS promoter plays, as iNOS, which is a key enzyme of oxidative stress, is activated by NF-κB. This study not only clarifies the pathogenesis of cataracts associated with diabetes, but it also contributes to the search for potential effective targets for the prevention and cure of diabetic cataracts. 
Acknowledgments
Supported by grants from the Major State Basic Research Development Program of China (973 Program) (2012CB518601); the National Natural Science Foundation of China (81070658); and the Hebei Natural Science Foundation (C2009001092). 
Disclosure: J. Jia, None; Y. Liu, None; X. Zhang, None; X. Liu, None; J. Qi, None 
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Figure 1
 
Changes in the level of NF-κB p65 at various glucose concentrations ( ± s). H2A.X was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; #compared with the 25 mM group, P < 0.05; n = 8.
Figure 1
 
Changes in the level of NF-κB p65 at various glucose concentrations ( ± s). H2A.X was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; #compared with the 25 mM group, P < 0.05; n = 8.
Figure 2
 
Levels of NF-κB p65 under high glucose (25 mM) and high glucose + PDTC conditions ( ± s). H2A.X was selected as an internal reference. *Compared with the 0 minute group, P < 0.05; △Compared with the 20 minute group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 2
 
Levels of NF-κB p65 under high glucose (25 mM) and high glucose + PDTC conditions ( ± s). H2A.X was selected as an internal reference. *Compared with the 0 minute group, P < 0.05; △Compared with the 20 minute group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 3
 
p65 nuclear translocation in high glucose conditions ( ± s). (A) Immunofluorescence imaging results: three white arrows indicate nuclei (blue), p65 (red), and merged results (purple), respectively. (B) Levels of p65 translocation after various incubation times. *Compared with the 0 minute group, P < 0.05; #compared with the 25 minute group, P < 0.05; n = 8.
Figure 3
 
p65 nuclear translocation in high glucose conditions ( ± s). (A) Immunofluorescence imaging results: three white arrows indicate nuclei (blue), p65 (red), and merged results (purple), respectively. (B) Levels of p65 translocation after various incubation times. *Compared with the 0 minute group, P < 0.05; #compared with the 25 minute group, P < 0.05; n = 8.
Figure 4
 
Changes in the level of iNOS mRNA at various glucose concentrations ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 4
 
Changes in the level of iNOS mRNA at various glucose concentrations ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 5
 
Changes in the level of iNOS protein at various glucose concentrations ( ± s). β-actin was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 5
 
Changes in the level of iNOS protein at various glucose concentrations ( ± s). β-actin was selected as an internal reference. *Compared with the 5.5 mM group, P < 0.05; n = 8.
Figure 6
 
Levels of iNOS mRNA under high glucose and high glucose + PDTC conditions ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 6
 
Levels of iNOS mRNA under high glucose and high glucose + PDTC conditions ( ± s). β-actin mRNA was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 7
 
Levels of iNOS protein under high glucose and high glucose + PDTC conditions ( ± s). β-actin was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 7
 
Levels of iNOS protein under high glucose and high glucose + PDTC conditions ( ± s). β-actin was selected as an internal reference. *Compared with the 0 hour high glucose group, P < 0.05; #compared with the high glucose + PDTC group at the same time point, P < 0.05; n = 8.
Figure 8
 
Results of ChIP performed on cells in the low glucose, high glucose, and high glucose + PDTC groups ( ± s). *Compared with the low glucose and high glucose + PDTC groups, P < 0.05; n = 8.
Figure 8
 
Results of ChIP performed on cells in the low glucose, high glucose, and high glucose + PDTC groups ( ± s). *Compared with the low glucose and high glucose + PDTC groups, P < 0.05; n = 8.
Table
 
Primer Sequences Employed for PCR
Table
 
Primer Sequences Employed for PCR
Gene Name Primer Sequences Product Size Annealing Temperature
iNOS Upstream 5′-CCTCGGCTCCAGCATGTACCCTCGG-3′ 135 bp 60°C
Downstream 5′-CGGAAGGCGTCCTCCTGCCCACTGA-3′
iNOS promoter Upstream 5′-CCACCCTTGGATACCGCAT-3′ 430 bp 53°C
Downstream 5′-CTCCCACTCCTACCCATTTCT-3′
β-actin Upstream 5′-TCGGGGCATCGGAACCGCTCA-3′ 404 bp 60°C
Downstream 5′-GAGACCTTCAAGACCCCAGCC-3′
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