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

首先，我们研究了活性氧在白细胞介素-8 （IL-8）基因激活中的作用，通过向人单核细胞系中添加几种抗氧化剂，脂多糖（LPS）刺激THP-1。转录因子NF-kappaB的激活对于lps诱导THP-1细胞中IL-8基因的激活是必不可少的，这在其他类型的细胞中也可以观察到。然而，当我们在完整的细胞中加入抗氧化剂时，我们没有观察到对IL-8基因激活和NF-kappaB激活的任何抑制。我们假设这些抗氧化剂的低渗透性可能是它们无法抑制IL-8基因激活的原因。因此，通过使用THP-1细胞的细胞提取物，我们开发了一种可以检测NF-kappaB的无细胞系统。在这个系统中，我们可以添加各种类型的抑制剂，而不考虑它们的渗透性或潜在的毒性。利用该系统，我们观察到NF-kappaB的抑制剂IkappaBalpha在NF-kappaB激活之前被磷酸化。此外，在该体系中没有观察到IkappaBalpha随后的降解。此外，两种不同类型的蛋白激酶(staurosporine-sensitive one （s)和酪氨酸激酶(s)）参与NF-kappaB的激活，而酪氨酸激酶抑制剂（而不是staurosporine）抑制ikappabα的磷酸化。为了阐明NF-kappaB活化的机制，我们试图对ikappabα磷酸化酶进行表征。在LPS的作用下，部分纯化的IkappaBalpha激酶迅速磷酸化了IkappaBalpha羧基末端酸性区域的丝氨酸和苏氨酸残基，特别是Ser293。该区域对应的肽在无细胞系统中抑制NF-kappaB激活以及IkappaBalpha磷酸化，表明该位点的磷酸化在这些过程中是不可或缺的。综上所述，这些结果表明LPS诱导IL-8基因激活的机制如下：LPS激活两种激酶，酪氨酸激酶(s)和staurosporine敏感激酶(s)。激活的酪氨酸激酶激活ikappabα激酶，进而使ikappabα羧基末端酸性区域的丝氨酸残基磷酸化。NF-kappaB与磷酸化的IkappaBalpha分离，从而转移到细胞核中并结合相应的顺式元件。staurosporin -sensitive kinase (s)可能通过磷酸化NF-kappaB的组分参与NF-kappaB的激活。在这些实验的同时，我们还分析了几种药物如FK506、糖皮质激素和干扰素α / β抑制IL-8基因的分子机制。我们观察到这些药物减弱NF-kappaB的激活，尽管每种药物的机制略有不同。这些结果提出了一种可能性，即靶向NF-kappaB活化的药物可能是一种很好的候选抗炎药，它可以抑制一种强效的中性粒细胞趋化细胞因子IL-8的产生。此外，我们的检测NF-kappaB激活的无细胞系统将有助于筛选这类药物。少

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