Reagents—Recombinant human IL-1β and -17A were purchased from R&D Systems Inc. (Minneapolis, MN). NF-κB p65 mouse IgG, p65 rabbit IgG, and IκB-α mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). NF-κB p50 mouse IgG was purchased from BioLegend (San Diego, CA). Nucleolin mouse IgG was purchased from Research Diagnostics Inc. (Concord, MA). β-Tubulin mouse IgG was purchased from Sigma-Aldrich. Actinomycin D was purchased from Calbiochem-Novabiochem.
Cell Culture and Cytokine Treatment
—HBE1 cell line is an immortalized line of normal human bronchial epithelial cells (38
). HBE1 cells were cultured on a plastic tissue culture surface in Ham's F-12/ Dulbecco's modified Eagle's medium (1:1) supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 μm
), cholera toxin (10 ng/ml), bovine hypothalamus extract (15 μg/ml), and bovine serum albumin (0.5 mg/ml). Recombinant human cytokines were dissolved in phosphate-buffered saline (PBS) with 1% bovine serum albumin and added directly to media of the culture (20 ng/ml). The control treatments had the same amount of PBS, 1% bovine serum albumin added. The primary TBE cell culture condition was described in the previous studies (1
). For the mRNA stability study, cells were pretreated with or without 20 ng/ml IL-17A for 24 h. Then 5 μg/ml actinomycin D was added to these cultures, which were harvested for RNA isolation at various hours as indicated.
Real Time Reverse Transcription-PCR Expression Analysis
—The RNA extraction, cDNA generation, primer sequences of glyceraldehyde-3-phosphate dehydrogenase, β-actin, and hBD-2
for real time PCR were described in our previous study (1
). The following PCR primers were used for human I
-ζ: forward, CATGGGAAATCCAATGAACAC; and reverse, GGCAACAGCAATATGAAGGAA.
NF-κB/Nuclear Extract Binding Enzyme-linked Immunosorbent Assay—BD™ TransFactor NF-κB Family Colorimetric kit from BD Biosciences Clontech was used to quantify the NF-κB-specific binding activity of nuclear extract. The quantification was carried out according to the manufacturer's protocol. Briefly to each well 20 μg of nuclear extract were added and incubated for 1 h at room temperature. Microtiter wells were then washed three times, and diluted primary antibodies against various NF-κB subunits were added (100 ml/well) and incubated further at room temperature for an hour. After extensive washing, diluted secondary antibody conjugated with horseradish peroxidase was added to each well and further incubated at room temperature for 30 min. After repeated washing, 100 μl of tetramethylbenzidine substrate solution were added to each well in the dark for the color development at room temperature. The reaction was quenched by 100 μl of 1 n HCl/well, and binding intensity was measured as absorbance at 450 nm using a microtiter plate reader.
Immunofluorescence Microscopy—HBE1 cells were plated to sterile Lab-Tek II chamber slides (Nalge Nunc International, Rochester, NY). At various times after IL-17A treatment, cells in slide chambers were fixed at 4 °C for overnight in PBS supplemented with 4% paraformaldehyde solution. The slide chambers were washed three times with PBS for 5 min each, permeabilized with 0.1% Triton X-100 in PBS for 30 min at 37 °C, and blocked with the blocking buffer containing 2% goat serum in PBS with Tween 20 for 30 min at 37 °C. The slide chambers were then stained with mouse anti-p50 monoclonal and rabbit anti-p65 polyclonal primary antibodies (1:200 dilution in blocking buffer) for 1 h at 37 °C, washed three times with PBS with Tween 20 for 5 min each, and incubated with fluorescently labeled Alexa Fluor 488-goat anti-mouse IgG and Alexa Fluor 568-goat anti-rabbit IgG secondary antibodies (1:500 dilution in blocking buffer) (Molecular Probes Inc., Eugene, OR) for 1 h at 37 °C. Nuclei were counterstained with VECTASHIELD® mounting medium with 4′,6′-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). The staining was visualized using a Zeiss AxioSkop fluorescence microscope (×40 objective).
Preparation of Nuclear Extracts—Nuclear lysates from cultured HBE1 cells were harvested according to the Panomics (Redwood City, CA) nuclear extraction protocol. In brief, HBE1 cells were lysed with a lysis buffer (10 mm HEPES (pH 7.9), 10 mm KCl, 10 mm EDTA, 1 mm dithiothreitol, 0.5% Igepal, and protease inhibitor mixture) on ice for 10 min and harvested using a sterile scraper. The cytosolic fraction was collected from the supernatant by centrifugation at 15,000 × g for 3 min at 4 °C. Pelleted nuclei were resuspended in extraction buffer (10 mm HEPES (pH 7.9), 400 mm NaCl, 1 mm EDTA, 10% glycerol, 1 mm dithiothreitol, and protease inhibitor mixture). After incubation at 4 °C for 2 h with gentle rocking, the nuclei were collected by centrifugation at 15,000 × g for 5 min at 4 °C. The resultant supernatants were collected and stored at -80 °C.
Western Blotting—HBE1 cells were treated with 20 ng/ml IL-17A and IL-1β, the positive control, at different time intervals. Cells were either harvested in radioimmune precipitation assay buffer followed by a brief sonication and centrifugation or with the Panomics Nuclear Extraction kit according to the manufacturer's protocol for nuclear and cytosolic protein extractions. The concentrations of the resulting total, nuclear, and cytosolic proteins were then determined by the Lowry method using bovine serum albumin as a standard. Twenty micrograms of protein extracts were subjected to 10% SDS-PAGE and blotted to polyvinylidene difluoride membrane for Western blot analysis.
Site-directed Mutagenesis of hBD-2 Promoter-Luciferase Reporter Plasmid and siRNA
-2210 promoter-luciferase reporter plasmid has been described in the previous study (1
). The three individual NF-κB sequences in the hBD-2
promoter construct, hBD-2
-2210/Luc, dκB(-2193 to -2182), pκB2 (-596 to -572), and pκB1 (-205 to -186), were mutagenized by using the Transformer™ site-directed mutagenesis kit (BD Biosciences Clontech). The resulting mutated NF-κB constructs were termed dκB-mut/Luc, pκB2-mut/Luc, pκB1-mut/Luc, and dκB+pκB2+pκB1mut/Luc (mutations on all three NF-κB sites). The authenticity of these mutations was confirmed by DNA sequencing. siRNAs against I
-ζ (identification number 33380) and random oligomer were purchased from Ambion (Austin, TX).
Transient Transfection and Luciferase Assay—HBE1 cells were seeded into 12-well plates at a density of 1 × 105 cells/well. One day after plating, cells were transfected with 0.5 μg of hBD-2-2210/Luc, dκB-mut/Luc, pκB2-mut/Luc, pκB1-mut/Luc, or dκB+pκB2+pκB1mut/Luc plasmid DNA and 50 ng of Renilla luciferase expression vector pRL-TK (Promega) using the FuGENE 6-based gene transfer protocol (Roche Diagnostics) according to the manufacturer's instructions. Eighteen hours after the transfection, cells were treated with 20 ng/ml IL-17A, and cell extracts were prepared for reporter gene assays 24 h after the IL-17A treatment. The reporter gene assays were carried out with the Dual-Glo™ Luciferase Assay System (Promega) according to the manufacturer's protocol. The relative hBD-2 promoter activities were expressed as relative luciferase units after normalization to the internal control, Renilla luciferase activity. The results were averaged from triplicate wells of three separate experiments. For siRNA transfection, cells were plated at 40–60% density a day before transfection. An Oligofectamine-based transfection kit (Invitrogen) was used according to the manufacturer's instruction. Sixteen hours after transfection, the siRNA transfection mixture was replaced with fresh culture medium. Two days later, cultures were depleted of hormonal supplements 24 h before IL-17A treatment. At various times after the treatment, cells were harvested for gene expression analyses.
NoShift p50 and p65 Binding Assay—The NoShift transcriptional factor assay kit (Novagen, Inc., Madison, WI) was used to measure binding of NF-κB p50 and p65 proteins to three NF-κB binding sequences on hBD-2 promoter as described in the manual. Briefly 10 μg each of sense and antisense 5′-biotinylated oligonucleotides (dκB-sense, 5′-CTTTGGGACTTCCCCAGCTA-3′;dκB-antisense, 5′-TAGCTGGGGAAGTCCCAAAG-3′;pκB2-sense, 5′-TGGGGAGTTTCAGGGGAACTTTCAC-3′, pκB2-antisense, 5′-GTGAAAGTTCCCCTGAAACTCCCCA-3′;pκB1-sense, 5′-AGGGATTTTCTGGGGTTTCC-3′, and pκB1-antisense, 5′-GGAAACCCCAGAAAATCCCT-3′) were dissolved to a final volume of 100 μl in a mixture of 0.5 m SSC (1× SSC is 0.15 m NaCl plus 0.015 m sodium citrate), 75 mm NaCl, and 7.5 mm sodium citrate (pH 7.0), heated to 100 °C (in boiling water bath) for 10 min, slowly cooled to room temperature, and then diluted to 10 pmol/μl. Nonbiotinylated oligonucleotides with the same sequence duplex that were used as competitor DNA were prepared in the same way except with a final concentration of 50 pmol/μl.
For measurement of binding affinity, the reaction mixtures mainly containing 1 pmol of biotinylated target DNA duplex, 20 μg of nuclear extract, and competitive nonbiotinylated DNA complexes were incubated on ice for 30 min. The reaction mixtures were then dispensed into freshly prepared streptavidin plates and incubated for 1 h at 37 °C. The binding of p50 and p65 was detected by incubation for 1 h at 37 °C with 100 μl of NF-κB p50 and p65 mouse IgG diluted 1:500 in NoShift antibody dilution buffer. After repeated washing of the plate, horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.) was added (1:1,000 dilution in NoShift antibody dilution buffer). After 30 min of incubation at 37 °C, wells were washed thoroughly, and tetramethylbenzidine substrate was added. The reaction was quenched by 100 μl of 1 n HCl/well, and binding intensity was measured as absorbance at 450 nm.
Chromatin Immunoprecipitation Assay—Formaldehyde cross-linking and chromatin immunoprecipitation in TBE cells were performed according to standard protocols on Farnham laboratory web site. After reversing the cross-links, the DNA was purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA) and analyzed using PCR amplification. Five microliters of DNA were used for PCR to detect the presence of specific DNA segments with the following primer pairs: dκB: forward, 5′-CATCCCCCAGTCTCTTCATCT-3′; reverse, 5′-ATGAGACCAGTGTCCAGGCTA-3′; pκB2: forward, 5′-GGTGTGAATGGAAGGAACTCA-3′, reverse, 5′-TTCAGCTCCTGGGGATGATAC-3′; and pκB1: forward, 5′-TGGCAGGTTATAGGTCCTGAG-3′; reverse, 5′-ATAAAGGTCCTGGTCCCTGGT-3′.
Statistical Analysis—Data are expressed as mean ± S.E. The number of repetitions for each experiment is given under “Results.” Paired comparisons were carried out by t test. Differences were considered significant for p values less than or equal to 0.05.