Fisetin is a selective adenosine triphosphate‐competitive inhibitor for mitogen‐activated protein kinase kinase 4 to inhibit lipopolysaccharide‐stimulated inflammation

Abstract

AbstractThe mitogen‐activated protein kinase kinase 4 (MKK4), a member of the MAP kinase kinase family, directly phosphorylates and activates the c‐Jun NH2‐terminal kinases (JNK), in response to proinflammatory cytokines and cellular stresses. Regulation of the MKK4 activity is considered to be a novel approach for the prevention and treatment of inflammation. The aim of this study was to identify whether fisetin, a potential anti‐inflammatory compound, targets MKK4‐JNK cascade to inhibit lipopolysaccharide (LPS)‐stimulated inflammatory response. RAW264 macrophage pretreated with fisetin following LPS stimulation was used as a cell model to investigate the transactivation and expression of related‐inflammatory genes by transient transfection assay, electrophoretic mobility shift assay (EMSA), or enzyme‐linked immunosorbent assay (ELISA), and cellular signaling as well as binding of related‐signal proteins by Western blot, pull‐down assay and kinase assay, and molecular modeling. The transactivation and expression of cyclooxygenase‐2 (COX‐2) gene as well as prostaglandin E2 (PGE2) secretion induced by LPS were inhibited by fisetin in a dose‐dependent manner. Signaling transduction analysis demonstrated that fisetin selectively inhibited MKK4‐JNK1/2 signaling to suppress the phosphorylation of transcription factor AP‐1 without affecting the NF‐κB and Jak2‐Stat3 signaling as well as the phosphorylation of Src, Syk, and TAK1. Furthermore, in vitro and ex vivo pull‐down assay using cell lysate or purified protein demonstrated that fisetin could bind directly to MKK4. Molecular modeling using the Molecular Operating Environment™ software indicated that fisetin docked into the ATP‐binding pocket of MKK4 with a binding energy of −71.75 kcal/mol and formed a 1.70 Å hydrogen bound with Asp247 residue of MKK4. The IC50 of fisetin against MKK4 was estimated as 2.899 μM in the kinase assay, and the ATP‐competitive effect was confirmed by ATP titration. Taken together, our data revealed that fisetin is a potent selective ATP‐competitive MKK4 inhibitor to suppress MKK4‐JNK1/2‐AP‐1 cascade for inhibiting LPS‐induced inflammation.