RNA was extracted from hTCEpi cells using RNA STAT 60 (Tel-TEST, Friendswood, TX). cDNA was generated from 2 μg of total RNA using Superscript First Round Synthesis with random primers (Invitrogen, Carlsbad, CA) according to manufacturer instructions. A 50 μl PCR reaction was performed as follows: 5 μmol/l each primer, 0.20 mM dNTPs, 5 μl of 10× PCR buffer (Sigma, St. Louis, MO), 7.5 mM MgCl2 (Sigma, St. Louis, MO), 1.0 μl Taq DNA Polymerase (5U/μl, Sigma, St. Louis, MO), and 2 μl cDNA. Gene specific primers were used to amplify the full length IGFBP3 coding sequence: forward 5′ ATGCAGCGGGCGCGAC 3′ and reverse 5′ CTACTTGCTCTGCATGCTGTAGCA 3′ with melting temperatures of 62.9°C and 59.2°C, respectively. The reaction conditions for PCR were an initial denaturation for one cycle at 94°C for 3 minutes, followed by 25 cycles at 94.0°C for 1 minute, 60°C for 45 seconds and 72.0°C for 45 seconds, and a final extension at 72.0°C for 7 minutes. Products were resolved on a 1.25% agarose gel stained with ethidium bromide and imaged on a Typhoon Variable Mode Imager.
Human donor tissue was obtained from Tissue Transplant Services, UT Southwestern Medical Center, Dallas, TX. All tissue was obtained fresh and processed within 6 hours of death. To establish the localization of IGFBP3 in the human corneal epithelium, non-fixed tissue was embedded in tissue embedding medium (Electron Microscopy Services, Hatfield, PA) and snap frozen in liquid nitrogen. 10 μm cryostat-sectioned tissue was permeabilized in cold acetone (-20°C), washed in phosphate buffered saline (PBS), and blocked in 10% donkey serum at 37°C for 30 minutes. Sections were incubated with a goat polyclonal antibody directed against the C-terminus of human IGFBP3 (10 μg/ml, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C, followed by incubation in 30 μg/ml FITC-conjugated secondary donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at 37°C for 2 hours. Tissue sections were counter-stained with 10 μg/μl Propidium Iodide (PI, Sigma, St. Louis, MO) to label all epithelial nuclei and mounted To confirm antibody specificity, the antibody was incubated in five fold excess of an available blocking peptide in 500 μl of PBS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 hours at 4°C with shaking. The primary antibody was omitted for a negative control. Sections were imaged on a Leica SP2 laser scanning confocal microscope (Leica Microsystems GmbH, Nussloch, Germany).
SDS PAGE and Western Blotting
Epithelial cell lysates were collected by lysing cells directly in plastic 6-well culture dishes using RIPA buffer containing a protease inhibitor cocktail tablet (Complete-Mini, Roche Diagnostics, Indianapolis, IN) on ice for 10 minutes. For collection of epithelial cells from tissue, an 8 mm circular trephine was used to separate cornea from limbus. Epithelial cells were removed by scraping using a sterile number 3 scalpel blade (Rotax International LTD, Sheffield, UK) and placed into RIPA/protease inhibitor cocktail. All lysates were snap frozen in liquid nitrogen and vortexed. For protein analysis from conditioned media, media was collected from T75 flasks after 48 hours and concentrated using iCON Protein Concentrators (Pierce, Rockford, IL). For analysis of resting, basal tears, tear samples were collected from the inferior tear meniscus at the lateral canthus of three non-dry eye patients using micro-pipettes (Drummond Scientific Co., Broomall, PA). Tear samples were added directly to 4× sample buffer. Cells transfected with pcDNA-IGFBP3, a plasmid encoding the cDNA for IGFBP3 (a gift of Dr. Sue Firth, the Kolling Institue, Sydney, Australia), and 0.5 μg rhIGFBP3 were used as a positive control. Transfections were performed using FuGene6 transfection reagent (Roche, Indianapolis, IN). All lysates and tear samples were boiled for 5 minutes in 4× sample buffer pH 6.8 containing 0.25 M tris, 8% lauryl sulfate, 40% glycerol, 20% mercaptoethanol and 0.04% bromophenol blue, resolved on a 12% SDS polyacrylamide gel and subsequently transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat milk and blotted using a 1:5000 dilution of a rabbit antiserum raised against rhIGFBP3 (Diagnostic Systems Laboratories, Webster, TX) overnight at 4°C. Following incubation in a 1:5000 dilution of an HRP-conjugated anti-rabbit IgG secondary antibody (Amersham Biosciences, Piscataway, NJ), protein was visualized using ECL Plus Detection Reagents (Amersham Biosciences, Piscataway, NJ) and imaged on a Typhoon Variable Mode Imager. Quantitative protein analysis was performed using Image Quant Software, Amersham Biosciences, Piscataway, NJ).
Cell Surface Association Assay
To determine if recombinant human IGFBP3 associated with the cell membrane and/or internalized, 2.2 × 105 cells/well were seeded onto plastic 6-well tissue culture dishes and treated with 500 ng/ml rhIGFBP3 for up to 48 hours. Media was removed and cells were washed thoroughly with PBS. Cells were then lysed using RIPA buffer as described above and analyzed by SDS PAGE and western blotting.
Real Time PCR
Real Time RT-PCR was used to assess total message levels for IGFBP3. hTCEpi cells were seeded onto plastic 6-well tissue culture dishes at a density of 2.2 × 105 cells/well and treated with 3.31 μM TSA for up to 30 hours to assess the ability of TSA to upregulate IGFBP3 or were allowed to grow over 7 days to assess IGFBP3 mRNA levels as a function of confluence. mRNA was extracted from hTCEpi cells by homogenization using the standard RNA STAT 60 protocol (TEL-TEST, Inc., Friendswood, TX). Following homogenization, RNA was extracted using chloroform, precipitated with isopropanol, washed in 75% ethanol and air-dried. RT PCR with First-Strand Synthesis using Random Primers (Invitrogen, Carlsbad, CA) was used to generate cDNA. Real time PCR was performed with a 20 μl reaction using 10 μl of 2× Taqman Universal Master Mix (Applied Biosystems, Foster City, CA), 900 nM forward/reverse primer, 200 nM probe, and 4 μl cDNA. Gene specific primers were used to amplify a 131 bp fragment for IGFBP3 (forward primer 5′ GGT GTC TGA TCC CAA GTT CC 3′ and reverse primer 5′ CGG AGG AGA AGT TCT GGG TA 3′). The hybridization probe was designed using Primer Express software (MGB-CGCTACAAAGTTGACTAC, Applied Biosystems, Foster City, CA). For quantitation of mRNA, a standard curve was prepared using serial dilutions of a known concentration of hTCEpi RNA. RNA concentration was determined by measuring the optical density at 260 nm on a Beckman DU 530 spectrophotometer and converted to μg/μl. A pre-designed assay reagent kit for β-actin was used as an endogenous control (Applied Biosystems, Foster City, CA), along with a no template control. All reactions were performed in duplicate in a 384 well plate using an ABI Prism 7900-HTA Sequence Detection System (Applied Biosystems). All assays were performed three independent times to allow for statistical analysis.
Annexin V Assay
To measure early apoptotic changes, 2.2 × 105 cells/dish were plated onto delta T4 tissue culture dishes and allowed to adhere for 24 hours. Cells were treated with either 500 ng/ml non-glycosylated rhIGFBP3 or 3.31 μM Trichostatin-A (TSA, Sigma, St. Louis, MO) for 0, 2, 4, 6, 8, 18, and 24 hours. Cells were washed once in PBS and followed by the addition of 1× binding buffer containing 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2. Cells were triple-labeled using an Annexin V Conjugate for Apoptosis Detection Alexa Fluor 647 (Molecular Probes, Invitrogen, Carlsbad, CA), 1 μg/ml PI, and 50 μM Calcein-AM (Molecular Probes) for 20 minutes at room temperature. Five regions of each culture dish were imaged on a Leica SP2 laser scanning confocal microscope. Cell counts were obtained using MetaMorph Software (Molecular Devices Corp., Downington, PA). The number of viable cells was determined by Calcein-AM staining. The percentage of Annexin V positive and PI positive cells was determined by dividing the number of positive cells by the total number of cells present in each field. All experiments were performed in duplicate and repeated twice.
To measure end stage apoptosis, TUNEL labeling was performed using the Apoptag Fluorescein In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA) according to manufacturer instructions. 5 × 104 hTCEpi cells per well were plated on collagen-coated coverslips and allowed to adhere for 24 hours. Cells were treated with either 3.3 μM TSA or 500 ng/ml rhIGFBP3 and collected at 0, 24, and 48 hours. Cells were fixed in 1% paraformaldehyde, washed with PBS, and post-fixed in 2:1 ethanol:acetic acid for 5 minutes at -20°C. Cells were washed in PBS and incubated in Equilibration buffer for 10 minutes at room temperature. Equilibration buffer was removed and cells were incubated in WS TdT Enzyme at 37°C for 1 hour, followed by washing with WS Stop/Wash buffer for 10 minutes at room temperature. Cells were washed again using PBS and then incubated in WS Anti-Digoxigenin Conjugate for 30 minutes at room temperature. Following a final wash in PBS, coverslips were mounted in a 50% (v/v) solution of PBS:glycerol containing 10 μg/μl PI, for visualization of epithelial cell nuclei. For a positive control, cells were incubated in DNase I (Invitrogen, Carlsbad, CA) for 20 minutes at room temperature prior to adding equilibration buffer. WS TdT enzyme was omitted in the negative control. Five regions of each culture dish were imaged on a Leica SP2 laser scanning confocal microscope. Cell counts were obtained using MetaMorph Software (Molecular Devices Corp., Downington, PA). The percentage of TUNEL positive cells was determined by dividing the number of positive cells by the total number of cells, assessed by PI staining, present in each field. All experiments were performed in duplicate and repeated twice.
Statistical analysis was performed using SigmaStat 3.1 (Systat Software, Inc., San Jose, CA). All data are expressed in mean ± standard deviation. A One-way Anaylsis of Variance was used to determine if a significant difference existed. The multiple comparison Student-Newman-Keuls Test was performed to assess a significant difference between groups. Statistical significance was set at P<0.05.