Material
RPMI 1640 medium with 2 mM glutamine was obtained from PAA Laboratories, Pasching, Austria. Sulforaphane (SFN) was obtained from Sigma-Aldrich Chemical Company, Munich, Germany. All other chemicals were of the highest quality commercially available.
Cell culture
The human synoviocyte cell line HSE was obtained from Oligene, Berlin, Germany. These cells were produced by immortalisation of primary human synovial fibroblasts from a confirmed RA patient. Immortalisation was performed using a pGEM vector containing a SV40 Tag-encoding DNA fragment [10]. The immortalised human synoviocyte cell line K4IM was a generous gift from Christian Kaps (Charité, Berlin, Germany). These cells originate from synovial tissue of a 41-year-old male donor suffering from a meniscus lesion and were also immortalised by a pGEM7/SV40 TAg vector construct. Several studies confirmed that both immortalised cell lines represent a valuable tool to study mechanisms that induce synoviocyte activation [11–13]. Both cell lines were cultured as monolayers in RPMI 1640 medium supplemented with 10% (v/v) foetal calf serum (FCS), 2 mM glutamine and 50 μg/mL penicillin-streptomycin.
Stimulation protocols
We used K4IM cells for the analysis of the inflammatory response of synoviocytes (Figures 1 and 2). The examination of matrix metalloproteinase (MMP) expression and activity was conducted with HSE cells (Figure 3), because it is known that these cells express MMP-3 and -9. For these experiments, cells were pre-treated for 30 min with solvent or increasing concentrations of SFN, before they were stimulated with 10 ng/mL TNF-α to examine the inhibitory effect of SFN on NF-κB, AP-1 and the downstream cytokine and MMP expression.
For experiments, which were performed to gain insights on the effects of SFN on already inflamed cells, the HSE cell line was used (Figures 4 and 5). These cells were pre-treated with 10 ng/mL TNF-α for 3 h to induce a proinflammatory state, before they were stimulated with 6.25 μM SFN for a further 6 h.
Transient transfection of cell lines
For transient co-transfection with reporter gene plasmids together with the Renilla luciferase coding phRL-TK internal control plasmid, 4.0 × 106 cells were seeded in a 100 mm dish one day before transfection. Transfection was carried out with Lipofectamine™ 2000 reagent (Invitrogen, Darmstadt, Germany) according to the manufacturer's recommendations. After removal of the transfection mixture, cells were trypsinised and 2.5 × 104 cells per well were seeded in a 96-well microtitre plate for stimulation experiments.
Stable Keap1 knockdown via lentiviral short hairpin RNA (shRNA) transduction
The real-time cell proliferation analysis (RTCA) experiment was performed with HSE cells, which were transduced with a pLKO.1 shRNA construct directed against Keap1 (MISSION™ shRNA assortment, Sigma-Aldrich, Germany) by lentiviral gene delivery. In order to exclude variations due to the vector backbone and to prove that observed effects are exclusively dependent on the shRNA, we also transduced HSE cells with a shNonTarget control construct.
Luciferase assays
After stimulation, cells were lysed in passive lysis buffer (Promega, Madison, WI, USA) and lysates were transferred to a white 96-well microtitre plate for luminescence measurement in a GloMax™ luminometer (Promega, Madison, WI, USA). Dual-luciferase reporter gene assay (Promega, Madison, WI, USA) was performed as described in Wruck et al. 2007. The following constructs were used:
p(NF- κ B)
3
-Luc refers to a concatenated trimer of the wild-type sequence ATGTGGGATTTTCCCATG (the core sequence is bold). Both strands:
5'-CATGTGGGATTTTCCCATGAGTGAGGGGACTTTCCCAGGCATGTGGGATTTTCCCATGG-3' and
5'-CTAGCCATGGGAAAATCCCACATGCCTGGGAAAGTCCCCTCACTCATGGGAAAATCCCACATGGTAC-3'.
pAP-1
5
-Luc refers to a concatenated pentamer of the AP-1 consensus sequence TGAGTCA.
pGL3-ARE refers to the antioxidant response element (ARE) consensus sequence of the rat NAD(P)H dehydrogenase, quinone 1 (NQO1) promoter region, as described in [14].
All strands were cloned in the pGL3-promoter vector (Promega, Madison, WI, USA). The luciferase assay was carried out as described in [15].
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-1β and IL-6 in the synoviocyte culture supernatants were determined by ELISA. The assay was performed according to the manufacturer's instructions.
Luminex xMAP™ multiplex assay
The total levels of secreted MMP-3 and MMP-9 were quantified by Luminex xMAP™ technology in duplicates. For that purpose, HSE culture supernatants were used after experimental treatment. The Fluorokine™ MAP human MMP base kit (Catalogue No. LMP000) was purchased from R&D Systems (Minneapolis, MN, USA) and utilised as recommended by the manufacturer.
Zymographic analysis
Zymographic analysis was used to determine the activity of MMP-9 after secretion into the cell culture medium over a fixed period of time. The method was performed according to a standard procedure described previously [16]. Briefly, following 24 h of experimental treatment, cell-conditioned media were harvested and concentrated for zymography. Total protein concentrations were determined by BCA (bicinchoninic acid) protein assay. Equal concentrations of total protein were used for analysis. Therefore, a total sample volume of 15 μL was loaded on a 12.5% PAGE gel containing 0.1% gelatin (BioRad, Munich, Germany). Gels were run for 90 min at 150 volts and were then incubated in 2.5% Triton-X (Merck, Darmstadt, Germany) for 1 h at room temperature (RT) on a rocker. In the next step, they were washed with distilled water and incubated overnight in renaturation buffer (10 mM Tris HCl, 1 mM CaCl2 × 2 H2O, 40 mM NaCl, 0.2 μM ZnCl2, pH 7.5) at 37°C. Gels were briefly washed and stained (1 g Coomassie blue, 200 mL methanol, 50 mL acetic acid, 250 mL H2O) for 1 h at RT on a rocker, followed by destaining in destaining buffer (200 mL methanol, 50 mL acetic acid, 250 mL H2O) until bands were clearly visible. Gels were scanned for densitometry analysis by ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). As MMP-2 is constitutively expressed and thus not regulated, its band served as an internal loading control for normalisation of densitometric data on MMP-9.
Cell viability assay
A 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assay was used to measure cell viability. Therefore the cells were seeded in 96-well plates. After treatment with or without TNF-α [10 ng/mL] for 3 h, cells were treated with 6.25 μM SFN for a further 6 h. After the stimulation, the media were supplemented with 10 μL/well (96-well plate) WST 2 h before spectrophotometric evaluation. Conversion of WST to formazan was measured at 450 nm by microplate spectrophotometry (Tecan Sunrise™, Männedorf, Switzerland). This reaction reflects the reductive capacity of the cells, thereby representing the viability.
Cytotoxicity assay
CytoTox-Glo™ cytotoxicity assay (Promega, Madison, WI, USA) was used to measure cytotoxicity. Cells were seeded in 96-well plates and after treatment with or without TNF-α [10 ng/mL] for 3 h, cells were treated with 6.25 μM SFN for a further 6 h. A CytoTox-Glo™ cytotoxicity kit was used according to the manufacturer's recommendation. Luminescence was measured in a 96-well plate reader (GloMax™ 96 microplate luminometer; Promega, Madison, WI, USA).
Quantification of caspase activities
For measurements of caspase 3 and 7 activity, the Caspase-Glo™ 3/7 assay (Promega, Madison, WI, USA) was carried out according to the manufacturer's instructions. Cells were treated as described for the WST and CytoTox-Glo™ assay. This assay is based on the cleavage of the DEVD sequence of a luminogenic substrate by caspases 3 and 7, which results in a luminescent signal.
Real-time cell analysis (RTCA)
We analysed the effect of Nrf2 and TNF-α on proliferation of HSE cells by continuous real-time impedance measurements using the xCELLigence system (Roche Diagnostics, Mannheim, Germany). The xCELLigence system contains a 96-well cell culture plate with a microelectrode array in the bottom of each well (E-Plate). The electrode impedance of the separate wells can be measured in defined intervals and is finally converted to a dimensionless parameter named Cell Index (CI). The CI is zero without cell adherence and increases when cells attach or proliferate on the microelectrode surface. To measure the background impedance all wells of the E-Plate were filled with 200 μL of growth medium. Afterwards 5.0 × 103 HSE cells stably expressing a shRNA targeting Keap1 or a non-targeting control shRNA were seeded by replacing 100 μL of the total medium per well. Subsequently, impedance measurements were conducted at intervals of 20 min under normal cell culture conditions (37°C in a humidified 5% CO2 atmosphere). After an overnight adherence phase, TNF-α was added to the respective wells in a final concentration of 10 ng/mL by replacing 100 μL medium per well, whereas control cells received equal amounts of fresh medium. Impedance measurements were continued then for additional 24 h. Afterwards the CI was normalised to the time-point of TNF-α addition and analysed using the RTCA software version 1.2.1 (Roche Diagnostics). All measurements were conducted with eight replicates.