Marienfeld Ralf, May Michael J, Berberich Ingolf, Serfling Edgar, Ghosh Sankar, Neumann Manfred
Section of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, Connecticut 06520, USA.
J Biol Chem. 2003 May 30;278(22):19852-60. doi: 10.1074/jbc.M301945200. Epub 2003 Mar 25.
RelB is an unusual member of the NF-kappaB transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-kappaB-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-kappaB1 or p52/NF-kappaB2, the precise molecular mechanisms through which it represses NF-kappaB remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-alpha-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA.RelB heterodimers that were unable to bind to kappaB sites in vitro strongly suggesting that these newly described NF-kappaB dimers cannot bind DNA. Expression pattern analysis revealed that RelA.RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IKKbeta. Functional characterization of RelA.RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by IkappaB proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA.RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-kappaB-dependent gene expression.
RelB是核因子κB转录因子家族中一个不同寻常的成员，它既是转录激活因子，也是核因子κB依赖性基因表达的抑制因子。尽管RelB与p50/核因子κB1或p52/核因子κB2结合时能促进基因表达，但其抑制核因子κB的确切分子机制仍不清楚。为了更详细地研究这种抑制功能，我们采用报告基因检测法，发现RelB在RelA水平发挥抑制作用。此外，电泳迁移率变动分析表明，体外翻译的RelB会损害RelA的DNA结合活性，而过表达的RelB会显著降低肿瘤坏死因子-α诱导的小鼠胚胎成纤维细胞中RelA的活性。有趣的是，这种抑制作用是由于形成了RelA.RelB异源二聚体，它们在体外无法与κB位点结合，这强烈表明这些新描述的核因子κB二聚体不能结合DNA。表达模式分析显示，RelA.RelB异源二聚体在淋巴细胞和非淋巴细胞中的水平相对较低。然而，在用佛波酯或脂多糖刺激后或通过组成型活性IKKβ过表达，这些复合物的含量会增加。对NIH3T3小鼠胚胎成纤维细胞中RelA.RelB异源二聚体的功能特性分析表明，它们不受IκB蛋白的调控，且位于细胞质和细胞核中。综上所述，我们的研究结果表明，RelA被隔离在转录无活性的RelA.RelB复合物中，这提供了一种分子机制，可能解释RelB对核因子κB依赖性基因表达的抑制作用。
