Compounds and Reagents.

PSTC, CDDO-Me, and the IκB kinase 2 (IKK2) inhibitor 5-(4-fluorophenyl)-2-ureidothiophene-3-carboxamide (Podolin et al., 2005) were synthesized by the Stress and Repair Discovery Performance Unit within the Respiratory Therapeutic Area at GlaxoSmithKline (GSK) (King of Prussia, PA). Sulforaphane (SFN) was purchased from Toronto Research Chemicals (Toronto, ON, Canada), and DMF was purchased from Vitas-M Laboratory (Champaign, IL).

Cells and Cell Culture.

Normal human bronchial epithelial (NHBE) cells were purchased and maintained in complete bronchial epithelial cell growth medium (BEGM) according to the manufacturer’s recommendations (Lonza, Walkersville, MD) and were used up to passage 5. BEAS-2B human epithelial cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in complete BEGM up to passage 10 for experimentation. Human embryonic kidney (HEK)-293 cells were purchased from American Type Culture Collection and were routinely cultured in Dulbecco’s modified Eagle’s medium containing high glucose, glutamine, and sodium pyruvate with 10% heat-inactivated fetal bovine serum purchased from Life Technologies (Grand Island, NY) and were used at passages 2–5 for experimentation. Epithelial cells derived from bronchial brushings of subjects with COPD (provided by Dr. Steven Kelsen, Center for Inflammation, Translational, and Clinical Lung Research Laboratory, Temple University, Philadelphia, PA; protocol number 11764 approved by the institutional review board at Temple University) were grown and maintained in a 1:1 mixture of bronchial epithelial cell basal medium (Lonza) and Dulbecco’s modified Eagle’s medium (Life Technologies) with all-trans retinoic acid (Sigma-Aldrich, St. Louis, MO) and the following additives from a BEGM SingleQuot kit (Lonza): human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, epinephrine, transferrin, insulin, triiodothyronine, gentamicin and amphotericin. Cells from subjects used in this study were grown at the air-liquid interface (ALI) at passage 2 on Transwell 24-well permeable membrane (Corning Inc., Corning, NY) submerged for the first 3 days, during which time the culture medium was changed on day 1 and every other day thereafter. The ALI was created once the cells reached 100% confluence, by removing the apical medium and restricting the culture feeding to the basolateral compartment of the plates. The culture medium was changed every other day until the cells were differentiated at approximately 28 days. The human biologic samples were sourced ethically and their research use was in accord with the terms of the informed consent. Studies were performed in accordance with the Declaration of Helsinki.

Transfections.

NHBE cells were plated at 5 × 10⁴ cells per well in 24-well plates and were transfected the following day with 25 nM nontargeting or Nrf2 small interfering RNA (siRNA) (Dharmacon/Thermo Scientific, Waltham, MA) for 48 hours according to the supplier’s general transfection protocol. The wells were aspirated and the cells were treated with vehicle [dimethylsulfoxide (DMSO); Sigma-Aldrich] or compound in complete bronchial epithelial cell basal media without antibiotics for 24 hours at 37°C and 5% CO₂. The wells were aspirated and the cells were lysed with Complete Lysis-M Buffer (Roche, Indianapolis, IN) on ice for protein analysis or lysis buffer from the Illustra RNA Kit (GE Healthcare Life Sciences, Pittsburgh, PA) for mRNA determination.

Western Blotting.

Whole-cell lysate protein concentrations were determined with a colorimetric assay detection kit (BioRad, Hercules, CA) and were subjected to immunoblot analysis. Equal amounts of denatured protein lysates were separated on a 12.5% Tris-HCl Criterion precast gel and were transferred to a nitrocellulose membrane (BioRad). The membrane was blocked with 5% (w/v) ECL Prime blocking agent (GE Healthcare Life Sciences, Piscataway, NJ) in phosphate-buffered saline (PBS) without Ca⁺² and Mg⁺² containing 0.1% (v/v) Tween 20 (Sigma-Aldrich). The blot was exposed to rabbit polyclonal anti-human heme oxygenase (HO)-1 (diluted 1:2000; Enzo Life Sciences, Farmingdale, NY) overnight at 4°C. Two PBS/0.1% Tween wash steps were performed followed by the addition of an anti-rabbit IgG horseradish peroxidase–conjugated secondary antibody (diluted 1:5000; Cell Signaling Technology, Danvers, MA). HO-1 protein levels were detected with ECL detection reagent (GE Healthcare Life Sciences) and the autoradiograph was developed with a processor (Konica, Tokyo, Japan). Protein loading was assessed by stripping the membrane and reprobing for β-actin using a goat polyclonal IgG anti-actin horseradish peroxidase–conjugated antibody (diluted 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Quantification of HO-1 and β-actin signals was performed using a BioRad Gel Doc EZ Imager and BioRad Image Laboratory Software (version 3.0).

Analysis of Gene Expression.

For human samples, RNA was isolated using the Illustra RNA Kit (GE Healthcare Life Sciences) and the TaqMan assay was performed using probe/primer pairs to detect human NQO1, HO-1, glutamate-cysteine ligase modifier subunit (GCLM), and thioredoxin reductase 1 (TXNRD1) (Applied Biosystems, Foster City, CA). Data were normalized to 18S levels using the ΔΔ threshold cycle (Ct) method. For rodent samples, RNA was isolated using TRIzol reagent (Life Technologies). Five-hundred nanograms of RNA from each sample was reverse transcribed in a 20-µl reaction using QuantiTect reverse transcription (Qiagen, Germantown, MD) according to recommended conditions. Real-time polymerase chain reaction was performed on the ABI ViiA7 real-time polymerase chain reaction system (Applied Biosystems) using 18S as the internal control. The cDNA was amplified with probe/primer pairs for mouse or rat Nqo1, Ho-1, sulfiredoxin 1 (Srxn1), and Txnrd1 (obtained from TaqMan gene expression assays; Applied Biosystems) using universal master mix (Applied Biosystems). An identical Ct was applied for each gene of interest. The comparative Ct (∆∆Ct) relative quantification method was used to calculate the relative mRNA levels of target genes as described in the Applied Biosystems Chemistry Guide.

For NanoString gene expression profiling, a set of Nrf2-dependent genes selected from the literature was used to generate a code set (NanoString Technologies, Seattle, WA) including four normalization controls (ACTB, GAPDH, GUSB, RPL13A). One-hundred nanograms of purified RNA (RNAspin Mini RNA Isolation Kit; GE Healthcare Life Sciences) was used for RNA quantification according to the manufacturer’s instructions. Data were analyzed using NanoString nSolver Analysis Software (version 1.1).

Nrf2 Nuclear Translocation.

NHBE cells were cultured on plastic as described above until they were >80% confluent. On the day of the assay, vehicle (DMSO), PSTC, or tert-butyl hydroquinone (tBHQ) (Crescent Chemical Co., Islandia, NY) was added for 6 hours and then nuclear extracts were generated using the protocol included with the TransAm Nrf2 kit (Active Motif, Carlsbad, CA). Protein concentrations were determined with a colorimetric assay detection kit (BioRad). Nuclear samples were then assayed using the TransAm Nrf2 kit. For siRNA studies, cells were transfected as described above for 48 hours followed by 6 hours of treatment with vehicle or compounds. Cells were then processed as described above.

NQO1 Enzyme Activity Assay.

NQO1-specific enzyme activity was measured using an assay adapted from Prochaska and Santamaria (1988). For human samples, NHBE cells were cultured in complete BEGM medium, treated with 25 nM Nrf2 or nontargeting siRNA for 48 hours where indicated, and then exposed to vehicle (DMSO) or compounds for 48 hours. The medium was removed and crude cellular lysates were made using cell lysis buffer (Cell Signaling Technology) containing a protease inhibitor tablet (EDTA-free complete mini protease inhibitor; Roche Life Science, Indianapolis, IN). An aliquot was removed for assaying protein content via a colorimetric assay detection kit (BioRad). NQO1 activity was determined by adding 40 μl sample to 200 μl NQO1 activity cocktail [25 mM Tris, pH 7.4, 0.067% bovine serum albumin, 0.01% Tween-20, 5 μM flavin adenine dinucleotide, 1 mM glucose-6-phosphate, 2 U/ml glucose-6-phosphate dehydrogenase, 3 μM NADP, 0.83 μM menadione, and 0.3 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in duplicate with 10 μl of 20 μM dicoumarol (Sigma-Aldrich) or water. Absorbance was then measured at 570 nm for 30 minutes. For rodent samples, lung tissue was homogenized in buffer (50 mM Tris-HCl, pH 7.5, 15 mM NaCl, 1% NP-40, 100 mM NaF, and 50 mM β-glycerophosphate) containing one protease inhibitor tablet (Roche Life Science) per 10 ml homogenization buffer. Homogenate was incubated on ice for 15 minutes and then centrifuged at 1000g for 15 minutes at 4°C. The entire sample was remixed and transferred to a clean 1.5-ml Eppendorf tube for centrifugation at 13,000g for 5 minutes at 4°C. Clear cytosolic supernatant was collected and used for determination of protein content (BioRad) and NQO1 activity. For the latter, 10 μl of 20 μM dicoumarol (Sigma-Aldrich) or water was added to 40 μl tissue lysates and then 200 μl NQO1 activity cocktail was added. Absorbance was measured at 570 nm for 30 minutes.

Glutathione Assay.

For human samples, NHBE cells at passage 4 were treated with vehicle (DMSO) or compounds for 18 hours followed by addition of tert-butyl hydroperoxide (tBHP) or N-acetylcysteine (Sigma-Aldrich) for 4 hours under the indicated conditions. Crude cell lysates were generated and assayed for glutathione content according to the manufacturer’s protocol using 5% sulfosalicylic acid (DetectX Glutathione Fluorescent Detection Kit; Arbor Assays, Ann Arbor, MI). For rat samples, glutathione content was determined following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). One-hundred milligrams of lung tissue was normalized to 0.10 mg/ml with 1× 2-(N-morpholino)ethanesulfonic acid buffer and homogenized using a gentleMACS Dissociator (Miltenyi Biotec Inc., Auburn, CA). Sample were centrifuged at 228g for 10 minutes at 4°C and then clarified at 13,000g for 5 minutes at 4°C. Samples were deproteinated at a 1:1 ratio with metaphosporic acid (500 μl sample plus 500 μl metaphosporic acid) and vortexed immediately. Fifty microliters of 4 M triethanolamine (Sigma-Aldrich) was then added to increase the pH, and samples were diluted at 1:5 and/or 1:10 for glutathione assay.

NF-κB Nuclear Translocation.

BEAS-2B cells were plated and treated with vehicle (DMSO) or compound for 30 minutes and then stimulated with 2 ng/ml interleukin (IL)-1β for 30 minutes. Cells were then fixed with 4% paraformaldehyde and NF-κB levels were determined using the NF-κB high content screening assay (Thermo Scientific) and imaging on a PerkinElmer Operetta system PerkinElmer (Waltham, MA). NF-κB levels were analyzed using a Harmony software (version 3.1) nuclear translocation script and are reported as the percent inhibition of the mean nuclear fraction of vehicle-treated controls. The IC₅₀ for each compound was determined using GraphPad Prism software (version 5.0; GraphPad Software Inc., La Jolla, CA) via nonlinear regression, using a sigmoidal dose-response (variable slope) and constraining the top to <100, the bottom to >0, and the Hill slope to <2.0.

Mechanistic Target of Rapamycin Assay.

HEK-293 cells were incubated for 1.5 hours with vehicle (DMSO), 20 nM rapamycin (Sigma-Aldrich), or Nrf2 activators at the indicated concentrations, followed by the addition of 100 nM insulin (Sigma-Aldrich) for 30 minutes. Cells were lysed in 0.2 ml lysis buffer and phospho-S6 ribosomal protein (Ser235/236) and total S6 ribosomal protein were analyzed by ELISA (Cell Signaling) per the manufacturer’s instructions. Values are expressed as the ratio of phospho-S6 optical density/total S6 optical density, normalized as the percentage of the average ratio with insulin.

Animals.

C57BL/6J mice (25–30 g; Jackson Laboratory, Bar Harbor, ME) and Han Wistar rats (290–370 g; Charles River Laboratories, Wilmington, MA) were maintained on a 12-hour/12-hour light/dark cycle and were provided Purina LabDiet 5001 (St. Louis, MO) and water ad libitum. All studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health and were reviewed and approved by the GSK Policy on the Care, Welfare, and Treatment of Laboratory Animals.

PSTC-Induced Gene Expression and NQO1 Activity, In Vivo.

To assess Nrf2-regulated mRNA expression and NQO1 enzyme activity after dosing of PSTC, seven to eight mice or five rats per treatment group received a single oral administration of vehicle [0.9% DMSO, 7% dimethylacetoacetamide (DMA; Sigma-Aldrich), 10% Cremophor El (Sigma-Aldrich), and 82.1% water] or PSTC at the indicated doses. Six and 48 hours after dosing, animals were euthanized (as described below) and lung tissue was collected for the analysis of gene expression and NQO1 enzyme activity, respectively, as described above. For gene expression analysis, 100–200 mg lung tissue was placed in 5 ml RNAlater (Thermo Scientific), the tissue was promptly cut into small 5-cm pieces, and the samples were stored at −80°C. For measurement of NQO1 activity, lung tissue was snap frozen and stored at −80°C.

Cigarette Smoke Exposure.

C57BL/6J mice received nose-only exposure to 4% cigarette smoke from 3R4F cigarettes (Reference Cigarette Program, College of Agriculture, University of Kentucky, Lexington, KY), for 2 hours per day for 3 days. Mainstream smoke was generated by a Baumgartner-Jaeger CSM 2070i smoking machine (CH Technologies Inc., Westwood, NJ), which contains a circular head that holds 30 cigarettes and performs 1 rpm. A 4% concentration of smoke was produced via a 2-second, 35-ml puff of smoke once per minute from each cigarette; 1 liter of the resulting smoke was mixed with 24 liters of air and delivered to the exposure tower. During exposure to smoke or air (sham controls), mice were maintained in restraining tubes containing stainless-steel nose cone inserts. Where indicated, mice received once-daily oral administration of vehicle (0.9% DMSO, 7% DMA, 10% Cremophor El, and 82.1% water) or compound during the 3 days of smoke exposure as well as 24 hours before the initial smoke exposure. Six or 24 hours after the final smoke exposure on day 3, mice were anesthetized with isoflurane and then euthanized via cardiac exsanguination followed by cervical dislocation. Bronchoalveolar lavage (BAL) fluid was collected for cell counts, or lung tissue was collected and 1) stored in RNAlater at −80°C for analysis of gene expression or 2) snap frozen and stored at −80°C for analysis of cytokine levels.

BAL Fluid Collection and Cell Counting.

For mice, the trachea was cannulated with a 3-inch piece of PE90 tubing (BD, Franklin Lakes, NJ) fitted over a blunt 21-gauge needle, to which a three-way stopcock was attached (Baxter Healthcare, Deerfield, IL). Four 1-ml aliquots of ice-cold PBS were injected and removed sequentially through the tubing. After centrifugation at 140g for 2 minutes, cell pellets from the four aliquots were combined. For rats, the trachea was cannulated with a gavage needle attached to a 5-ml syringe. One 3-ml aliquot of ice-cold PBS was injected and removed through the gavage needle, and the resulting BAL fluid was centrifuged at 140g for 2 minutes. Total cells were counted using a hemocytometer. Differential cell analysis was performed on cytospins using Wright-Giemsa stain.

Cytokine Analysis.

Lung tissue was weighed, suspended in 1 ml PBS/g tissue, and homogenized using a gentleMACS Octo Dissociator with M tubes (Miltenyi Biotec Inc., Auburn, CA). The tissue homogenate was then centrifuged at 2500g for 10 minutes, and the resulting supernatant was centrifuged at 400g for 10 minutes. Keratinocyte-derived cytokine (KC; CXCL1) levels were measured using a mouse ELISA kit purchased from R&D Systems (Minneapolis, MN). Assays were performed according to the manufacturer’s instructions.

Ozone Exposure.

Han Wistar rats were exposed to ozone generated by an Oxycycler ozonator (Biospherix Inc., Lacona, NY), in which compressed air and oxygen were passed through the ozonator and flowed into a plexiglass chamber containing the animals. Twenty-four hours prior to ozone injury, rats received a single oral administration of vehicle (0.9% DMSO, 7% DMA, 10% Cremophor El, and 82.1% water) or compound. Rats were exposed to 1 ppm ozone for a 3-hour period. Ozone concentrations, humidity, and CO₂ within the chamber were constantly monitored using Oxycycler Watview software and an independent ozone monitor (Teledyne Advanced Pollution Instrumentation Inc., Thousand Oaks, CA). The rats were euthanized 15 minutes after injury by intraperitoneal administration of 1 ml Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI). BAL fluid was collected for cell counts, or lung tissue was collected, snap frozen, and stored at −80°C for analysis of glutathione content.

Quantitative Analysis of PSTC and the Corresponding Hydroxy Acid in Blood.

The blood samples were immediately added to acidified acetonitrile, 0.1% formic acid, containing 75 ng/ml propranolol as the internal standard for mass spectrometry analysis, in a 1:4 ratio (i.e., 50 µl blood into 200 µl acetonitrile/formic acid/propranolol) and stored in a −80°C freezer until analysis. For analysis of drug concentration, the blood samples were thawed, vortexed, and centrifuged (1207g, 4°C, 10 minutes). A 50-µl aliquot of the supernatant was vortexed with 50 µl water for injection onto the mass spectrometer. Analysis of the samples was performed using liquid chromatography/tandem mass spectrometric detection. Twenty-microliter injections were made onto the liquid chromatography/tandem mass spectrometric system using Agilent 1100 Series binary pumps (Agilent Technologies, Santa Clara, CA) and a CTC PAL Autosampler (CTC Analytics, Zwingen, Switzerland). The mobile phase consisted of a gradient that transitioned linearly from 95% aqueous 10 mM ammonium formate, pH 3, and 5% acetonitrile to 66% acetonitrile over 2 minutes (500 μl/min flow rate) and then to 95% acetonitrile over an additional minute, was held at those conditions for 0.5 minutes, and then reconditioned back to initial conditions for an additional 2.5 minutes. An Agilent SB-C18 4.6- × 50-mm analytical column was used to retain the analytes. The eluent flowed into a Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems) using negative-ion turbo ion spray multiple-reaction monitoring for the first 3.35 minutes and then switching to positive-ion turbo ion spray multiple-reaction monitoring for 2.65 minutes. PSTC was characterized by the transition of the mass-to-charge ratio (m/z) 356.1 parent (M + H)⁺ ion to its m/z 213.1 product ion, generated at optimized collision energy. The internal standard, propranolol, was characterized by the transition of the m/z 260.2 parent (M + H)⁺ ion to its m/z 116.1 product ion, generated at optimized collision energy. The corresponding hydroxy acid of PSTC was characterized by the transition of the m/z 372.0 (M − H)⁻ ion to its m/z 328.2 ion.

Statistical Analysis.

Data are presented as the mean ± S.E.M. Statistical significance was determined using a one-way analysis of variance with a Dunnett or Bonferroni post-test. P values of ≤0.05 were considered significant.