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

Research Project

Project/Area Number 06670346
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Immunology
Research InstitutionKanazawa University

Principal Investigator

MUKAIDA Naofumi  Cancer Research Institute Associate Professor, がん研究所, 助教授 (30182067)

Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
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)
Keywordsinterleukin-8 / NF-kappaB / IkappaBalpha / phosphorylation / tyrosine kinase / lipopolysaccharide / serine / threonine kinase / IkBα / NF-κB / IκB / リン酸化酵素 / 抗酸化剤
Research Abstract

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

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (33 results)

All Other

All Publications (33 results)

  • [Publications] Okamoto,S.-i.: "The interleukin-8 AP-1 and kB-like sites are genetic end targets of FK506-sensitive pathway accompanied with calcium mobilization." J.Biol.Chem.269. 8582-8589 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Mukaida,N.: "Novel mechanism of glucocortiocid-mediated gene repression : unclear factor kB is target for glucocortiocid-mediated interleukin 8 gene repression." J.Biol.Chem.269. 13289-13295 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Oliveira,I.C.: "Transcriptional inhibition of the interleukin-8 gene by interferon is mediated by the NF-kB site." Mol.Cell.Biol.14. 5300-5308 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ishikawa,Y.: "Establishment of lipopolysaccharide-dependent nuclear factor-kB activation in a cell-free system." J.Biol.Chem.270. 4158-4164 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuno,K.: "Identification of an IkBa-associated protein kinese in a human monocyte cell line and detemination of its phosphorylation sites of lkBa." J.Biol.Chem.270. 27914-27919 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Mukaida,N.: "Molecular msechanism of interleukin8 (IL-8) gene expression." J.Leukocyte Biol.56. 554-558 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Matsushima,K.: "Proceeding of lllrd International Mochida Memorial Symposium" The Mochida Memorial Foundation for Medical and Pharmaceutical Research, 11 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto,S.: "In the Chemokines Proceeding of lllrd International Symposium of Chemotactic Cytokines" Plenum, 6 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto, S.-i.: "The interleukin-8 AP-1 and kB-like sites are genetic end targets of FK506-sensitive pathway accompanied with calcium mobilization." J Biol. Chem.269. 8582-8589 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Mukaida, N.: "Novel mechanism of glucocortiocid-mediated gene repression : nuclear factor kB is target for glucocortiocid-mediated interleukin 8 gene repression." J.Biol. Chem.269. 13289-13295 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Oliveira, I.C.: "Transcriptional inhibition of the interleukin-8 gene by interferon is mediated by the NF-kB site." Mol. Cell. Biol.14. 5300-5308 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ishikawa, Y.: "Establishment of lipopolysaccharide-dependent nuclear factor-kB activation in a cell-free system." J.Biol. Chem.270. 4158-4164 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kuno, K.: "Identification of an IkBa-associated protein kinese in a human monocyte cell line and determination of its phosphorylation sites of IkBa." J.Biol. Chem.270. 27914-27919 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Mukaida, N.: "Molecular msechanism of interleukin8 (IL-8) gene expression." J.Leukocyte Biol.56. 554-558 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto, S.-i.: "The proceeding of IIIrd Intl.Symp. Chemotactic Cytokines." Plenum. 7. (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Matsushima, K.: "Proceeding of IIIrd International Mochida Memorial Symposium" The Mochida Memorial Foundation for Medical and Pharmaceutical Researcdh, Tokyo. 8. (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Okamoto,S-i.: "The interleukin-8 AP-1 and kB-like sites are genetic end targets of FK506-sensitive pathway accompanied with calcium mobilization." J. Biol. Chem.269. 8582-8589 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] Mukaida,N.: "Novel mechanism of qlucocortiocid-mediated gene repression:nuclear factor kB is target for glucocortiocid-mediated interleukin 8 gene repression." J. Biol. Chem.269. 13289-13295 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Oliveira,I.C.: "Transcriptional inhibition of the interleukin-8 gene by interferon is mediated by the NF-kB site." Mol. Cell. Biol.14. 5300-5308 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ishikawa,Y.: "Establishment of lipopolysaccharide-dependent nuclear factor-kB activation in a cell-free system." J. Biol. Chem.270. 4158-4164 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kuno,K.: "Identification of an IkBa-associated protein kinese in a human monocyte cell line and determination of its phosphorylation sites of IkBa." J. Biol. Chem.270. 27914-27919 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] Mukaida,N.: "Molecular msechanism of interleukin8(IL-8)gene expression." J. Leukocyte Biol.56. 554-558 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] Matsushima,K.: "Proceeding of IIIrd International Mochida Memorial Symposium" The Mochida Memorial Foundation for Medical and Pharmaceutical Research, 11 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] Okamoto,S.: "In the Chemokines Proceeding of IIIrd International Symposium of Chemotactic Cytokines" Plenum, 6 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] Harada,A.et.al.: "Cloning of cDNA encoding a mouse homolog of the interleukin 8 receptor." Gene. 142. 297-300 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Oliveira,I.C.et.al.: "Transcriptional inhibition of the interleukin 8 gene by interferon is mediated" Mol.Cell Biol.14. 5300-5308 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Okamoto,S.et.al.: "The interleukin-8 AP-1 and kB-like sites are genetic end target of FK506-sensitive pathway" J.Biol.Chem.269. 8582-8589 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Mukaida,N.et.al.: "Molecular mechanism of interleukin 8 gene expression." J.Leukocyte Biology. 56. 554-558 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Yasumoto,K.et.al.: "Molecular analysis of cytokine network involbed in causing cachexia in colon 26" Cancer Res.55. 921-927 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Ishikawa,Y.: "Establishment of lipopolysaccharide-dependent nulear factor-kB activation in a cell-free system." J.Biol.Chem.270. 4158-4164 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Mukaida,N.et.al.: "Novel Mechanism of Glucocorticoid-mediated Gene Repression:nuclear factor-kB is target for glucocorticoid-mediated interleukin 8 gene repression." J.Biol.Chem.269. 13289-13295 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Wada,T.et.al.: "Prevention of proteinuria by the administration of anti-interleukin 8 antibody in experimental acute immune complex-induced glomerulonephritis." J.Exp.Med.180. 1135-1140 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Murayama,T.et.al.: "Enhancement of human cytomegalovirus replication in human lung fibroblast cell line by interleukin 8." J.Virol.68. 7582-7585 (1994)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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