Volume 119, Issue 5, Pages 1209-1218 (November 2000) Suppression of NF-κB activity by sulfasalazine is mediated by direct inhibition of IκB kinases α and β  Christoph K. Weber, Susanne Liptay, Thomas Wirth, Guido Adler, Roland M. Schmid  Gastroenterology  Volume 119, Issue 5, Pages 1209-1218 (November 2000) DOI: 10.1053/gast.2000.19458 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Sulfasalazine inhibits nuclear NF-κB/Rel and transcriptional activity induced by TNF-α or TPA. (A) Electromobility shift assay showing inhibition of TNF-α–induced NF-κB/Rel binding activity by sulfasalazine. Jurkat T cells were left untreated (lane 1) or stimulated with TNF-α (150 U/mL for 30 minutes) (lanes 2–6). Cells were pretreated for 1 or 3 hours with 2 mmol/L sulfasalazine before stimulation with TNF-α (lanes 3 and 4). Binding specificity was confirmed by incubation with a 100-fold of an unrelated oligonucleotide spanning an Sp1 binding site (lane 6). The arrows indicate the position of the κB-specific DNA binding activity and the position of a nonspecific (n.s.) activity. (B) Jurkat T cells or (C and D) SW620 colon cells were transfected with a κB-dependent reporter construct (3×IgκBLuc). After 18 hours, cultures were treated with sulfasalazine (0.1, 1, or 2 mmol/L) 30 minutes before stimulation with (B and C) 150 U/mL TNF-α or (D) 10 ng/mL TPA. Extracts were prepared after an additional 18 hours and assayed for luciferase activity. Luciferase activity obtained without adding sulfasalazine was set to 100%. Values are expressed as means ± SEM of at least 3 independent experiments performed in duplicate. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Sulfasalazine inhibits NIK-induced κB-dependent transcription. Jurkat T cells (■) and SW620 colon cells (▩) were transfected with 3×IgκBLuc and 200 ng of a NIK expression vector and incubated after 18 hours with either medium alone (co) or with sulfasalazine (SS), 5-ASA, 4-ASA, or sulfapyridine (SP) at 2 mmol/L. After another 18 hours, luciferase activity was determined. Values are expressed as means ± SEM of at least 3 independent experiments performed in duplicate. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Sulfasalazine, but not 4-ASA, 5-ASA, or sulfapyridine, blocks IKK-α– and IKK-β–induced NF-κB/Rel–dependent transcription. (A) Jurkat T cells were transfected with a reporter construct (3×IgκBLuc) together with an empty expression vector or increasing amounts of an HA-IKK-α expression vector as indicated. (B) Jurkat T cells were similarly transfected with an HA-IKK-β expression vector. (C) For inhibition experiments, Jurkat T cells (■) or SW620 cells (▩) were transfected with 3×IgκBLuc and 5 mg of HA-IKK-α expression vector and incubated for 18 hours with either medium alone (co) or with sulfasalazine (SS), 5-ASA, 4-ASA, or sulfapyridine (SP) for another 18 hours. (D) In a similar experiment, cells were transfected with the 3×lgκBLuc reporter construct and 5 mg of HA-IKK-β expression vector and treated after 18 hours as indicated. After another 18 hours, luciferase activity was determined. Values are expressed as means ± SEM of at least 3 independent experiments performed in duplicate. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Sulfasalazine, but not 4-ASA, 5-ASA, or sulfapyridine, inhibits endogenous IKK activity. (A–C) SW620 cells or (D–F) Jurkat T cells were treated for 30 minutes with medium alone, sulfasalazine (0.5, 1, or 2 mmol/L), sulfapyridine (2 mmol/L), 5-ASA (2 mmol/L), or 4-ASA (2 mmol/L) before stimulation with TNF-α, 150 U/mL for 30 minutes. (A and D) IKK-α and IKK-β were immunopurified from cell extracts, and kinase activity was determined using GST-IκBα (1-54) as substrate. (B and E) Immunoprecipitates were analyzed by Western blotting using an IKK antibody recognizing IKK-α and IKK-β. (C and F) Kinase activity was quantitated using a phosphorimager and plotted as percent activity. Findings are representative of 3 independent experiments. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Sulfasalazine does not inhibit TNF-α–induced phosphorylation of Erk1/2, JNK1, or p38. As indicated, SW620 colonic cells were pretreated with medium containing 2 mmol/L sulfasalazine for 30 minutes, then stimulated with TNF-α (150 U/mL for 10 minutes) or left untreated. Activated (A) Erk1/2, (B) JNK1, or (C) p38 was detected by Western blotting using phospho-specific antibodies (upper panel). To determine the total amount of kinases, membranes were stripped and reprobed with antibodies against wild-type kinases. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Sulfasalazine inhibits activity and autophosphorylation of purified IKK-α and IKK-β in vitro. Recombinant (A) IKK-α and (B) IKK-β were incubated with DMSO (control) or the indicated concentrations of sulfasalazine in the presence of [γ-32P]ATP and GST-IκBα (1-54) for 30 minutes. Proteins were separated by SDS-PAGE. The upper panel shows representative autoradiographs of (A) IKK-α and (B) IKK-β autophosphorylation as well as substrate phosphorylation. *GST-IκBα bands were quantitated and plotted (lower panel). Values are expressed as means ± SEM of at least 3 independent experiments. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Sulfasalazine, but not 5-ASA, sulfapyridine, or aspirin, inhibits IKK-α/IKK-β activity. Recombinant (A) IKK-α and (B) IKK-β were incubated with DMSO, sulfasalazine, 5-ASA, sulfapyridine, or aspirin at 1 mmol/L in the presence of [γ-32P]ATP and GST-IκBα (1-54) for 30 minutes. Thereafter, proteins were separated by SDS-PAGE (upper panel). GST-IκBα bands were cut out, and activity was determined in a scintillation counter (lower panel). Values are expressed as means ± SEM of at least 3 independent experiments. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Sulfasalazine-mediated inhibition of IKK-α and IKK-β activity is blocked by increasing the ATP concentration. Recombinant (A) IKK-α and (B) IKK-β were incubated with the indicated concentrations of sulfasalazine and ATP. Kinase reaction was initiated by adding recombinant GST-IκBα (1-54) as substrate. Parallel kinase reactions were performed without the inhibitor. After 30 minutes, kinase reactions were stopped and proteins were separated by SDS-PAGE. GST-IκBα (1-54) bands were cut out, and activity was determined in a scintillation counter. Values obtained from kinase reactions with sulfasalazine without the addition of ATP were set to 100%. Inhibition of IKK activity by sulfasalazine is expressed as the mean ± SEM of at least 3 independent experiments. Gastroenterology 2000 119, 1209-1218DOI: (10.1053/gast.2000.19458) Copyright © 2000 American Gastroenterological Association Terms and Conditions