Vanden Berghe W, Francesconi E, De Bosscher K, Resche-Rigon M, Haegeman G
Department of Molecular Biology University of Gent and Flanders Interuniversity Institute for Biotechnology, Gent, Belgium.
Mol Pharmacol. 1999 Oct;56(4):797-806.
Synthetic glucocorticoids (GCs) remain among the most effective agents for the management of chronic inflammatory diseases. However, major side effects severely limit their therapeutic use. Physiologic and therapeutic activities of GCs are mediated by a nuclear receptor belonging to a superfamily of ligand-inducible transcription factors that, in addition to directly regulating their cognate gene programs, can also mutually interfere with other signaling pathways. We recently identified selective ligands of the glucocorticoid receptor that dissociate transactivation from activator protein 1 transrepression, and most importantly retain in vivo anti-inflammatory activity. To further document the mechanisms of action sustaining the observed in vivo activity, we report here on the interference of dissociated GCs with nuclear factor kappaB (NF-kappaB)-driven gene activation. We show that dissociated GCs repress tumor necrosis factor-induced interleukin-6 gene expression by an NF-kappaB-dependent mechanism, without changing the expression level of inhibitor kappaB. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with the various compounds. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines constitutively expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB, which was able to efficiently repress a Gal4-dependent luciferase reporter gene upon addition of the dissociated compounds. We therefore conclude that, in addition to dissociating transactivation from activator protein 1 transrepression, dissociated GCs mediate inhibition of NF-kappaB signaling by a mechanism that is independent of inhibitor kappaB induction.
合成糖皮质激素（GCs）仍然是治疗慢性炎症性疾病最有效的药物之一。然而，严重的副作用极大地限制了它们的治疗应用。GCs的生理和治疗活性由一种核受体介导，该核受体属于配体诱导转录因子超家族，除了直接调节其同源基因程序外，还能相互干扰其他信号通路。我们最近鉴定出了糖皮质激素受体的选择性配体，它们能使反式激活与激活蛋白1反式抑制解离，最重要的是保留了体内抗炎活性。为了进一步证明维持观察到的体内活性的作用机制，我们在此报告解离的GCs对核因子κB（NF-κB）驱动的基因激活的干扰。我们发现，解离的GCs通过NF-κB依赖性机制抑制肿瘤坏死因子诱导的白细胞介素-6基因表达，而不改变抑制因子κB的表达水平。在用各种化合物刺激细胞后，诱导的NF-κB的DNA结合活性也保持不变。通过分析组成性表达酵母Gal4蛋白的DNA结合结构域与NF-κB的反式激活p65亚基之间融合蛋白的细胞系，获得了直接核作用机制的证据，在加入解离的化合物后，该细胞系能够有效抑制Gal4依赖性荧光素酶报告基因。因此，我们得出结论，除了使反式激活与激活蛋白1反式抑制解离外，解离的GCs还通过一种独立于抑制因子κB诱导的机制介导对NF-κB信号的抑制。
