Suppression of NF-kappaB activity by sulfasalazine is mediated by direct inhibition of IkappaB kinases alpha and beta.
OBJECTIVE
Activation of NF-kappaB/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-kappaB/Rel signaling. The aim of this study was to define the molecular mechanism by which sulfasalazine inhibits NF-kappaB activation.


METHODS
The effects of sulfasalazine and its moieties on NF-kappaB signaling were evaluated using electromobility shift, transfection, and immune complex kinase assays. The direct effect of sulfasalazine on IkappaB kinase (IKK) activity was investigated using purified recombinant IKK-alpha and -beta proteins.


RESULTS
NF-kappaB/Rel activity induced by tumor necrosis factor alpha, 12-O-tetradecanoylphorbol-13-acetate, or overexpression of NF-kappaB-inducing kinase, IKK-alpha, IKK-beta, or constitutively active IKK-alpha and IKK-beta mutants was inhibited dose dependently by sulfasalazine. Sulfasalazine inhibited tumor necrosis factor alpha-induced activation of endogenous IKK in Jurkat T cells and SW620 colon cells, as well as the catalytic activity of purified IKK-alpha and IKK-beta 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-alpha and -beta 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-alpha and -beta by antagonizing adenosine triphosphate binding. The suppression of NF-kappaB activation by inhibition of the IKKs contributes to the well-known anti-inflammatory and immunosuppressive effects of sulfasalazine.
Weber
C K
CK
Department of Internal Medicine I, University of Ulm, Ulm, Germany.
Liptay
S
S
Wirth
T
T
Adler
G
G
Schmid
R M
RM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002460:Cell Line; D004338:Drug Combinations; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005765:Gastrointestinal Agents / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism; D019169:Jurkat Cells; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D012460:Sulfasalazine / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000097782:NF-kappaB-Inducing Kinase
D000255:Adenosine Triphosphate; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D005765:Gastrointestinal Agents; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D012460:Sulfasalazine; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human