Gastroenterology

Gastroenterology

Volume 119, Issue 5, November 2000, Pages 1209-1218
Gastroenterology

Alimentary Tract
Suppression of NF-κB activity by sulfasalazine is mediated by direct inhibition of IκB kinases α and β,☆☆

https://doi.org/10.1053/gast.2000.19458Get rights and content

Abstract

Background & Aims: Activation of NF-κB/Rel has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Various drugs used in the treatment of IBD, such as glucocorticoids, 5-aminosalicylic acid, and sulfasalazine, interfere with NF-κB/Rel signaling. The aim of this study was to define the molecular mechanism by which sulfasalazine inhibits NF-κB activation. Methods: The effects of sulfasalazine and its moieties on NF-κB signaling were evaluated using electromobility shift, transfection, and immune complex kinase assays. The direct effect of sulfasalazine on IκB kinase (IKK) activity was investigated using purified recombinant IKK-α and -β proteins. Results: NF-κB/Rel activity induced by tumor necrosis factor α, 12-O-tetradecanoylphorbol-13-acetate, or overexpression of NF-κB–inducing kinase, IKK-α, IKK-β, or constitutively active IKK-α and IKK-β mutants was inhibited dose dependently by sulfasalazine. Sulfasalazine inhibited tumor necrosis factor α–induced activation of endogenous IKK in Jurkat T cells and SW620 colon cells, as well as the catalytic activity of purified IKK-α and IKK-β in vitro. In contrast, the moieties of sulfasalazine, 5-aminosalicylic acid, and sulfapyridine or 4-aminosalicylic acid had no effect. Activation of extracellular signal-related kinase (ERK) 1 and 2, c-Jun-N-terminal kinase (JNK) 1, and p38 was unaffected by sulfasalazine. The decrease in substrate phosphorylation by IKK-α and -β is associated with a decrease in autophosphorylation of IKKs and can be antagonized by excess adenosine triphosphate. Conclusions: These data identify sulfasalazine as a direct inhibitor of IKK-α and -β by antagonizing adenosine triphosphate binding. The suppression of NF-κB activation by inhibition of the IKKs contributes to the well-known anti-inflammatory and immunosuppressive effects of sulfasalazine.

GASTROENTEROLOGY 2000;119:1209-1218

Section snippets

Cell culture and treatments

Jurkat T cells were grown in RPMI, and SW620 colonic epithelial cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 2 mmol/L glutamine, and 1% (wt/vol) penicillin/streptomycin. Recombinant human TNF-α, TPA, sulfasalazine, 4-aminosalicylic acid (4-ASA), 5-aminosalicylic acid (5-ASA), sulfapyridine, and acetylsalicylic acid (aspirin, ASA) were purchased from Sigma (Deisenhofen, Germany). Sulfasalazine, 4-ASA, 5-ASA, sulfapyridine, and aspirin were

Inhibition of NF-κB/Rel activation and κB-dependent transcription in jurkat T cells

NF-κB/Rel activity of Jurkat T cells induced by TNF-α was analyzed by electrophoretic mobility shift assay in the presence or absence of sulfasalazine (Figure 1A).

. 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

Discussion

Most of the beneficial effects of salicylates such as sulfasalazine are attributed to their inhibition of cyclooxygenase and prostaglandin H synthase; however, prostaglandin-independent effects, such as inhibition of the transcription factor NF-κB/Rel, have been shown.10 We previously demonstrated that sulfasalazine inhibits NF-κB/Rel activation induced by TNF-α, lipopolysaccharide, and TPA in a colonic epithelial cell line.13 The molecular mechanism for this inhibition remained unclear. In

Acknowledgements

The authors thank Esther Rüber and Sabine Schirmer for excellent technical assistance; Sonja Aigner for help in preparing the manuscript; Michael Karin (San Diego, California) for plasmids GST-IκBα (1-54) and GST-IκBα (1-54,S32A,S36A) and expression vectors for HA-IKK-α, HA-IKK-α, HA-IKK-EEα, HA-IKK-EEβ, and NIK; J. Li (Department of Biology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT) for recombinant IKK-α and IKK-β purified from Sf9 cells; and Joseph Slupsky for critical reading of

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    Address requests for reprints to: Roland M. Schmid, M.D., Department of Internal Medicine I, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany. e-mail: [email protected]; fax: (49) 731-50-24302.

    ☆☆

    Supported in part by grants from the Novartis-Stiftung für Therapeutische Forschung and Deutsche Krebshilfe (to R.M.S. and S.L.).

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