Abstract：
The 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.
摘要：
丝裂原活化蛋白激酶激酶4(MKK4),MAP激酶激酶家族的成员，直接磷酸化并激活c-JunNH2末端激酶（JNK），对促炎细胞因子和细胞应激的反应。MKK4活性的调节被认为是预防和治疗炎症的新方法。这项研究的目的是确定非瑟酮是否，一种潜在的抗炎化合物，靶向MKK4-JNK级联以抑制脂多糖(LPS)刺激的炎症反应。使用LPS刺激后用fisetin预处理的RAW264巨噬细胞作为细胞模型，通过瞬时转染试验研究相关炎症基因的反式激活和表达。电泳迁移率变动分析(EMSA)，或酶联免疫吸附测定（ELISA），和细胞信号以及通过蛋白质印迹结合相关信号蛋白，下拉测定和激酶测定，和分子建模。非塞素以剂量依赖性方式抑制LPS诱导的环氧合酶-2（COX-2）基因的反式激活和表达以及前列腺素E2（PGE2）的分泌。信号传导分析表明，fisetin选择性抑制MKK4-JNK1/2信号传导以抑制转录因子AP-1的磷酸化，而不影响NF-κB和Jak2-Stat3信号传导以及Src的磷酸化，Syk,和TAK1。此外，使用细胞裂解物或纯化的蛋白质的体外和离体下拉测定表明，非瑟酮可以直接结合MKK4。使用MolecularOperatingEnvironment™软件进行的分子建模表明，fisetin以-71.75kcal/mol的结合能停靠在MKK4的ATP结合袋中，并形成了与MKK4的Asp247残基结合的1.70µ氢。非塞素对MKK4的IC50在激酶测定中估计为2.899μM，并通过ATP滴定证实了ATP竞争效应。一起来看,我们的数据显示,Fisetin是一种有效的选择性ATP竞争性MKK4抑制剂,可以抑制MKK4-JNK1/2-AP-1级联,从而抑制LPS诱导的炎症.
