Mechanisms for regulation of activation of innate immunity by a nuclear protein, IκB-ζ which functions in both acceleration and inhibition of transcription

我们分析了IκB-ζ的分子功能、转录调控机制和刺激特异性诱导机制，这些机制在促进和抑制炎症基因的转录方面发挥了作用。首先，我们在IκB-ζ的第153-157位氨基酸中发现了一个核定位信号，在第329-402位氨基酸中发现了一个具有转录激活活性的区域。此外，我们还发现，在脂多糖刺激下，IκB-ζ的过表达在转录水平上抑制了肿瘤坏死因子-α的产生，而加速了白细胞介素6的产生。对I-κB-ζ基因突变小鼠的分析表明，IκB-ζ缺陷的巨噬细胞对内毒素的反应产生正常水平的肿瘤坏死因子-α和一氧化氮，而IL-6的产生被取消。在体内，内毒素处理后的IL-12的产生受到抑制，而肿瘤坏死因子-α的产生显著增加。其次，对被IκB-ζ加速转录的基因的启动子分析表明，启动子中的NF-κB结合位点对于IκB-ζ介导的转录激活是必不可少的，但不是充分的，激活需要另一个顺式元件。因此，顺式元件的存在决定了IκB-ζ在每个启动子上的功能。第三，关于刺激特异性诱导IκB-ζ的机制，我们发现IκB-ζ的稳定性在内毒素或IL-1β刺激下显著上调，但不受α刺激的影响。此外，我们还发现，IκB-ζ基因3‘非翻译区的一个序列通过转录后水平决定了IκB-ζ的刺激特异性诱导。因此，I-κB-ζ是调节炎症反应的关键分子之一，炎症反应是在天然免疫刺激下特异性诱导的，在抑制另一亚组基因表达的同时，对一组炎症基因的表达是必不可少的。

We analyzed molecular functions, mechanisms for transcriptional regulation, and mechanisms for stimuli-specific induction of IκB-ζ, which function in both acceleration and inhibition of transcription of genes on inflammation. Firstly, we identified a nuclear localization signal in a region of amino acids 153-157 of IκB-ζ and a domain with transcriptional activation activity in a region of amino acids 329-402. Furthermore, we found that overexpression of IκB-ζ resulted in inhibition of tumor necrosis factor (TNF)-α production and acceleration of interleukin (IL)-6 production, on transcriptional level, upon stimulation with lipopolysaccharide (LPS). Analyses on IκB-ζ gene-disrupted mice revealed that IκB-ζ-deficient macrophages produced normal levels of TNF-α and nitric oxide in response to LPS, but IL-6 production was abolished, and that, in vivo, IL-12 production upon treatment with LPS was impaired whereas TNF-α production was strongly augmented. Secondly, promoter analyses of genes whose transcription is accelerated by IκB-ζ showed that NF-κB binding sites in the promoter is essential for the IκB-ζ-mediated transcriptional activation, but not sufficient and another cis-element is required for the activation. Thus, the presence of the cis-element determines the function of IκB-ζ on each promoter. Thirdly, regarding the mechanisms for stimuli-specific induction of IκB-ζ, we found that stability of IκB-ζ mRNA is strongly up-regulated upon stimulation with LPS or IL- 1β, but not with TNF-α. Furthermore, we found that a sequence in the 3'-untranslated region of IκB-ζ mRNA determines the stimuli-specific induction of IκB-ζ by post-transcriptional level. Therefore, IκB-ζ is one of key players in regulation of inflammatory responses, which is specifically induced upon stimulation of innate immunity, and is essential for the expression of a subset of inflammatory genes while it inhibits the expression of another subset of genes.