Cell culture and Materials
Human lens epithelial cells (HLE B3), propagated and immortalized as previously described (Andley et al. 1994
) were generously provided by Dr. Usha Andley (Washington University, St. Louis, MO). These cells were grown in MEM medium supplemented with 20% fetal calf serum and 50 μg/ml gentamycin in 100 × 20 mm tissue culture plates in humid atmosphere with 5% CO2
at 37°C. Cells reached confluence with 6 × 106
cells / plate in 4 days. Fresh eyes from 6-8 months old pigs were obtained from Farmland Inc (Crete, NE). Human recombinant anti TBP-2 mouse monoclonal antibody was purchased from MBL international Corporation (Woburn, MA). All the other chemicals and reagents were standard commercial products of analytical grade.
Enzyme and other assays
Protein concentrations were determined by BCA following manufacture's protocol (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as the standard. The activity of Trx was determined by a previously described method (Holmgren and Bjornstedt, 1995
). Hydrogen peroxide concentration in lens organ culture medium was measured following Hildebrant et al. (Hildebrant et al., 1978
Recombinant human lens TRx and anti- TRx antibody
Human cDNA for TRx was cloned into pET23a(+) vector, expressed in E. coli
, and the TRx was purified using His-bind column as described earlier (Yegorova et al., 2003
). Rabbit anti-TRx antibody was prepared by Proteintech Group, Inc. (Chicago, IL, USA) using purified recombinant human TRx. Antiserum was made by immunization of albino rabbit first with subcutaneous injection of 1 mg of recombinant human TRx (emulsified with Freund's complete adjuvant), followed by second injection ( 1mg TRx in Freund's incomplete adjuvant) eight weeks later. Antiserum was collected ten days after the booster and affinity purified by a TRx affinity column, prepared by linking TRx to agarose with cyanogen bromide.
Cloning of human lens TBP-2
To clone human lens TBP-2 cDNA, PCR was carried out with cDNA prepared from the total RNA extracted from HLE-B3 cells as a template. Primers were designed to clone TBP-2 into Escherichia coli expression system (forward primer 5'GAATTCGATGGT GATGTTCAAGAAGATC3'; reverse primer 5'CGCTCGAGTCACTGACAATTGTT GTTGA3'). Both primers were designed based on the known nucleotide sequence of human brain TBP-2 sequence (GenBank accession number NM_006472). The conditions of the PCR were: initial 94°C for 2 min., 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The obtained PCR fragments were separated on a 2% (wt/vol) agarose gel, and the band corresponding to 1176 bp were isolated and purified using a gel extraction kit (Qiagen, Valencia, CA). The purified PCR fragment were cloned downstream of the cytomegalovirus (CMV) promoter into PCR 3.1-Uni Vector (Invitrogen, San Diego, CA) and used to transform TOP 10F′ cells (Invitrogen). The transformants were selected on Luria-Bertani (LB)-coated plates with 50 μg/mL kanamycin. The recombinant plasmids designated as pCR-TBP-2 were analyzed for the presence and orientation of the insert by restriction enzymes Xho1 and EcoR1. Sequencing of the TBP-2 insert was performed and analyzed at the DNA Sequencing Facility the University of Nebraska-Lincoln (Lincoln, NE). The sequence was compared with published TBP-2 sequences using NCBI Blast, vector NTI, and GCG programs.
TBP-2 protein expression and purification
Human lens TBP-2 was expressed in E.coli by using pET-His expression system from Novagen (Madison, WI). To clone TBP-2 cDNA fragment into pET 28a(+) vector, primers were modified (forward 5'ACGCGTGCCATG GTG ATG TTC AAG AAG ATC3', reverse 5' CCATCGATTCACTGCACATTGTTGTTGAG 3') by introducing Mul1 and Cla1 restriction sites. The PCR product was then cloned into pET28a (+) between Mul1 and ClaI sites, and the positive expression plasmid pET-TBP-2(His) was used to transform into the BL21 (DE3) E.coli cells (Novagen). For the expression of recombinant TBP-2 protein, transformed BL21 (DE3) cells were grown at 37°C in LB medium with 100 μg/ml kanamycine until the absorbance at 600 nm reached to 0.4-0.6. The cells were induced for TBP-2 expression by 1mM isopropyl-1-thio-β-Dgalactopyranoside (IPTG) for 3-4 hrs and then harvested by centrifugation at 6,000 rpm for 30 min. The cell pellets were resuspended in 30 ml lysis buffer (BugBuster with Benzonaze Nuclease; Novagen), incubated for 20 min at room temperature, followed by centrifugation at 16,000 rpm for 45 min. The precipitate with the inclusion body fraction of the lyzed cells was used to purify TBP-2 using His Bind column (Novagen) according to the manufacturer's protocol. The size and purity of recombinant TBP-2 protein was confirmed by SDS-PAGE and the identity of the protein was confirmed by protein sequencing (Protein sequencing facility, University of Nebraska, Lincoln).
Immunoprecipitation of TBP-2-TRx complex by anti-TRx and anti-tbp-2 antibodies
Both anti-TBP-2 monoclonal antibody and anti-TRx antibodies were used for the immunoprecipitation of TRx-TBP-2 complex in vivo. HLE B3 cell lysate was incubated for 3 hrs at 4°C either with 10 μl (2 μg) of anti-TRx antibodies or with 50 μl (50 μg) of anti-TBP-2 antibodies, followed by adding 20 μl Protein-A Agarose beads (Santa Cruz, CA) for overnight incubation at 4°C. Immunoprecipitate was collected by centrifugation at 2,500 rpm for 5 min at 4°C, washed 4 times with ice-cold washing buffer (150 mM NaCl, 1% Tween 20, 1% deoxycholate, and 20 mM Tris HCL pH 7.5), and then resuspended in 40 μl of 1X electrophoresis sample buffer. Seize® X Protein A Immunoprecipitation kit (PIERCE, IL) was used to immunoprecipitate TRx-TBP-2 complex according to manufacture's protocol. These immunoprecipitates, which were freed from antibodies used for the immunoprecipitation were then used for Western blot analysis.
Effect of H2O2 on TBP-2 expression in HLE B3 cells
HLE B3 cells (4.2 x 106) were used for the study. Prior to H2O2 treatment, the cells were gradually serum-starved by incubating overnight in MEM with 2% FBS and then in serum-free MEM for another 30 min before subjecting to a bolus of 0.1 mM H2O2 for 0, 5, 10, 15, 20, and 30 min. At each time point medium was removed for analysis of H2O2 concentration. Cell lysates were made with 500 μL of lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 (vol/vol), 1 mM Na3VO4, 0.25% Na-deoxycholate, 1 mM PMSF, 1 mM NaF, and 1 μg/mL each of aprotinin, pepstatin, and leupeptin) and used for immunoblot analysis for TBP-2 and TRx.
Effect of H2O2 on TBP-2 and TRx protein expression in cultured pig lenses
Fresh pig lenses were surgically removed from the eye balls and immediately pre-incubated in 4 ml TC-199 medium for 2 hrs at 37°C in a CO2 incubator. The lenses were exposed to medium with and without 0.2 mM H2O2 (0.1 mM H2O2 and 2.31 units of glucose oxidase for final and constant H2O2 level) for 0, 2, 4, 6, 9, 12, 18, and 24 hrs with 3 lenses per each time point. Fifty μl of the medium was removed at each time interval and used for H2O2 assay. At the same time points, three lenses were removed, rinsed with PBS, epithelial layers peeled off surgically and immediately frozen on dry ice. The pooled three epithelial layers were 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, 1μg/ml aprotinin, and 100 μg/ml PMSF using Polytron glass hand homogenizer. The homogenate was centrifuged at 13,000 g for 25 min at 4° C, and the supernatant was used for immunoblot analysis.
Extraction of Total RNA and detection of mRNA for TRx and TBP-2 by RT-PCR
In a separate experiment, H2O2-treated and the control untreated pig lenses were used for RT-PCR analysis. The epithelial layers from each time point were peeled off as described above and immediately transferred to RNAlater (QIAGEN Inc., Valencia, CA 91355) RNA stabilization reagent and kept submerged at room temperature for 6 hrs. The total mRNA was then extracted using RNeasy RNA extraction mini kit (QIAGEN), following the manufacturer's protocol. Aliquots containing equal amounts (1μg) of mRNA were reverse transcribed using cloned AMV first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA 92008). Following primers were designed and synthesized to detect TRx, TBP-2 and β-actin cDNA, respectively. TRx: (Forward) 5'-GCTGCCAAGATGGTG AA GCAGATT-3', (Reverse) 5'-GCAACATCCTGACAGTCATCCACA-3'; TBP-2: (Forward) 5'-GAATTCGATGGTGATGTTCAAGAAGATC3' (Reverse) 5'-CGCTCGA GTCACTGACAATTGTTGTTGA3'; β-actin: (Forward) 5'-GTGGGGCGCCCAG GCACCA-3', (Reverse) 5'-CTCCTTAATGTCACGCAGGATTTC-3'. The sizes for the amplification products of TRx, TBP-2, and β-actin were 206 bp, 1176 bp, and 420 bp, respectively. Equal amounts of synthesized cDNA were amplified by PCR using Taq DNA polymerase (Invitrogen) to detect mRNA for TRx, TBP-2 and β-actin. The PCR conditions used were 94° C for 1 min, 50° C for 1 min and 72° C for 1 min and 35 cycles. Aliquots were taken from PCR mixtures and analyzed by 1% agarose gel electrophoresis.
Total mRNA was extracted from HLE B3 cells. cDNA was synthesized from 50ng of total RNA with AMV First-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CL) according to the manufacturer's instructions. Primers and FAM-labeled probes for human TRx, TBP-2 and β-actin were obtained from Applied Biosystems (Branchburg, NJ). Quantitative real-time PCR (QPCR) was then performed using a real-time detection system called Icycler IQ (Bio-Rad, Hercules, CL). Briefly; reactions were performed in 96-well optical reaction plates of 25 μl volume, containing cDNA equivalent to 50ng of DNase-digested RNA,12.5 μl of TaqMan Universal Master mixture and optimized concentrations of forward and reverse primers. The mRNA expression of TRx and TBP-2 were normalized to the level of GAPDH mRNA as described previously (Livak and Schmittgen 2001
Western Blot analysis
Tissue or cell homogenates were separated by 10% SDS-PAGE, transferred to a membrane (TransBlot, Bio-Rad), and probed with either anti-TRx, or anti-TBP-2 antibodies diluted in TBST buffer (10 mM Tris-HCl [ph 7.5], 100 mM NaCl and 0.1 % Tween 20), followed by treatment with either goat anti-rabbit IgG-horseradish peroxidase (for membrane probed with rabbit anti human Trx) or goat anti-mouse IgG2b-horseradish Peroxidase (for membranes probed with mouse anti human TBP-2 antibody). Immunodetection was performed with chemiluminescent reagent (Santa Cruz Biotechnology). The immunoblot was analyzed with an imaging system (Fluor-S MAX MultImager, Bio-Rad, Richmond, CA). Densitometric analyses of immunoblots were carried out using an image processing and analysis program, Scion Image (Scion Corporation, Frederick, MD).
Over-expression of TBP-2 in HLE-B3 cells
Sense cDNA for TBP-2 was introduced into the multi-cloning site of Geneticin (G418 sulfate)-resistant mammalian expression vector pcDNA3.1 (+) to construct plasmids. The plasmids were then transfected into HLE-B3 cells using Lipofectamine plus reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Cells transfected with the cDNA were incubated with transfection medium for one day and the cells were passaged into new plates with fresh culture medium. The cells were grown for two days before transferring to fresh medium containing 1 mg/ml geneticin for selection and the cells were fed every four days. After 4 weeks of selection, the cells were passaged into new plates containing fresh medium with 400 μg/ml geneticin and grown to 50-60% confluency before use.
To investigate the proliferation of control and TBP-2 over-expressed HLE B3 cells, 1×105 cells were plated in 6 well plates, and the number of cells was counted by using a haemacytometer. The assay was performed over a period of 4 days.
Effect of H2O2 on apoptosis of control and TBP-2 over-expressed HLE-B3 cells
Control and TBP-2 over-expressing HLE B3 cells were cultured in MEM with 20% FBS in 60 mm dishes. When cells were 50-60 % confluent, the serum-containing medium was removed and cells were washed two times with PBS before exposing to serum-free medium with and without 0.1 mM H2O2 for 1 hr. The cells were then placed in fresh MEM with 20% FBS, incubated for another 16 hrs followed by analysis for apoptosis. To quantify apoptosis Annexin V apoptosis detection kit was used (Biovision, Mountain View, CA). The H2O2 pre-treated cells were trypsinized and washed with 20% FBS containing MEM once and then once with PBS. Then the cells were stained with Annexin V-FITC according to manufacturer's protocol and analyzed by flow cytometry (FACScan flow cytometer; Becton Dickinson, Franklin Lakes, NJ). Excitation was set at 558 nm and the emission was measured at 530 nm in 10,000 gated cells using linear amplification. The arithmetic mean fluorescence channel was derived by CellQuest® software (Becton Dickinson, NJ).