Molecular biological analysis of mechanisms of interleukin-8 production, with a refernce to the roles of reactive oxygen

参考活性氧的作用，对 IL-8 产生机制进行分子生物学分析

基本信息

批准号：
06670346
负责人：
MUKAIDA Naofumi
金额：
$ 1.41万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

First, we investigated the roles of reactive oxygen species in interleukin-8 (IL-8) gene activation by adding several anti-oxidants to human monocytic cell line, THP-1 stimulated with lipopolysaccahride (LPS). The activation of a transcription factor, NF-kappaB,was indispensable for LPS-induced IL-8 gene activation in THP-1 cells, as observed on other types of cells. However, when we added anti-oxidants to intact cells, we failed to observe any inhibition on IL-8 gene activation as well as NF-kappaB activation.We postulated that low-permeability of these anti-oxidants might account for their failure to inhibit IL-8 gene activation. Thus, by using cell extracts from THP-1 cells, we developed a cell-free system where NF-kappaB can be detected. In this system, we can add various types of inhibitors without considering their permeability or potential toxicity. Using this system, we observed that IkappaBalpha, an inhibitor of NF-kappaB,was phosphorylated before NF-kappaB activation. Moreove … More r, the subsequent degradation of IkappaBalpha was not observed in this system. Furthermore, two distinct types of protein kinases, staurosporine-sensitive one (s) and tyrosine kinase (s), were involved in NF-kappaB activation whereas tyrosine kinase inhibitors but not staurosporine inhibited the phosphorylation of IkappaBalpha.In order to clarify the mechanism of NF-kappaB activation, we tried to characterize the IkappaBalpha phosphorylase. In response to LPS,partially purified IkappaBalpha kinase rapidly phosphorylated serine and threonine residues present in the carboxy-terminal acidic region of IkappaBalpha, particularly, Ser293. The peptide corresponding to this region inhibited NF-kappaB activation as well as IkappaBalpha phosphorylation in a cell-free system, indicating that the phosphorylation of this site is indispensable for these processes. Collectively, these results suggest the mechanisms of LPS-induced IL-8 gene activation as follows :LPS activates two types of kinases, tyrosine kinase (s) and staurosporine-sensitive one (s). Activated tyrosine kinase activates IkappaBalpha kinase, which in turn phosphorylates mainly serine residues present in the carboxy-terminal acidic region of IkappaBalpha. NF-kappaB dissociate from phosphoryalted IkappaBalpha, thereby translocating into nucleus and binding the corresponding cis-element. Staurosporine-sensitive kinase (s) may participate in NF-kappaB activation by phosphorylating the components of NF-kappaB.In paralled with these experiments, we also analyzed the molecular mechanisms of IL-8 gene repression by several agents such as FK506, a glucocorticoid, and interferon alpha/beta. We observed that these agents attenuated the NF-kappaB activation although the mechanisms were slightly different from each agent. These results raised the possibilities that the drug targeting NF-kappaB activation may be a good candidate for an anti-inflammatory agent by inhibiting the production of a potent neutrophil chemotactic cytokine, IL-8. Furthermore, our cell-free system to detect NF-kappaB activation will be useful for screening this kind of agents. Less

首先，我们通过在人单核细胞系中添加几种抗氧化剂，研究了活性氧中的活性氧在白介素8（IL-8）基因激活中的作用，该细胞细胞系被脂肪polysaccahride（LPS）刺激的THP-1。在THP-1细胞中LPS诱导的IL-8基因活化是必不可少的，如在其他类型的细胞上，对于LPS诱导的IL-8基因激活是必不可少的。但是，当我们在完整细胞中添加抗氧化剂时，我们无法观察到对IL-8基因激活以及NF-kappab激活的任何抑制作用。通过使用THP-1细胞中的细胞提取物，我们开发了一个无细胞的系统，可以检测到NF-kappab。在该系统中，我们可以在不考虑其渗透性或潜在毒性的情况下添加各种类型的抑制剂。使用该系统，我们观察到NF-kappab的抑制剂Ikappabalpha在NF-kappab激活之前被磷酸化。 Moreove…更多的R，在该系统中未观察到Ikappabalpha的随后降解。此外，NF-kappab激活参与了两种不同类型的蛋白激酶，星形孢菌素敏感的一种和酪氨酸激酶，而酪氨酸激酶抑制剂，但不是staurosporine抑制剂。为了响应LP，部分纯化的Ikappabalpha激酶快速磷酸化的丝氨酸和苏氨酸保留在Ikappabalpha的羧基末端酸性区域中，尤其是SER293。该肽对应于该区域抑制了无细胞系统中的NF-kappab激活以及ikappabalpha磷酸化，这表明该位点的磷酸化对于这些过程是必不可少的。总的来说，这些结果表明LPS诱导的IL-8基因激活的机制如下：LPS激活两种类型的激酶，酪氨酸激酶（S）和符号孢菌素敏感的一种。活化的酪氨酸激酶激活了Ikappabalpha激酶，进而将丝氨酸磷酸化主要保留在Ikappabalpha的羧基末端酸性区域中。 NF-kappab与磷酸化的ikappabalpha分离，从而易位成核并结合相应的顺式元素。与这些实验并行的NF-kappab的成分，可以通过磷酸化NF-kappab的激活来参与NF-kappab的激活，我们还分析了几种Ass fk506（例如FK506），一种glucoforoidoinoid alpha和Interstra和Interstra和Internerpha，我们还分析了IL-8基因表达的分子机制。我们观察到这些试剂减弱了NF-kappab的激活，尽管这些机制与每个药物略有不同。这些结果提高了靶向NF-kappab激活的药物可能通过抑制潜在的中性粒细胞趋化细胞因子IL-8的产生而成为抗炎剂的良好候选者。此外，我们检测NF-kappab激活的无细胞系统将有助于筛选这种药物。较少的