|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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
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