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Regulation of NF-κB in Human Vascular Tissue

  • Nandor Marczin
  • Ginette Hoare
  • Karen Morrison
  • Adrian Chester
  • Magdi Yacoub
Part of the NATO ASI Series book series (NSSA, volume 294)

Abstract

Activation of NF-κB transcription factor may contribute to proliferative and inflammatory responses of vascular smooth muscle (SMC) during atherogenesis and systemic inflammatory response. This study was designed to elucidate the cellular mechanisms underlying cytokine-induced NF-κB activation in human aortic SMC. The inhibitor (I-κB) and the DNA binding p65 subunit of NF-κB were identified in the cytoplasm of unstimulated cells by immunocytochemistry and Western blotting. Exposure to increasing concentrations of IL-1β (1–100 U/ml), TNFα (10–1000 U/ml) or bacterial lipopolysaccharide (10–1000 ng/ml) caused increased phosphorylation of I-κB followed by complete depletion of I-κB within 30 minutes. Depletion of I-κB was associated with increased nuclear content of p65. To investigate the role of oxidant stress in cytokine-induced NF-κB activation, SMC were pretreated with antioxidants N-acetyl-L-cysteine (NAC) or metal chelator pyrrolidine-dithio-carbamate (PDTC). PDTC (20mM) but not NAC (20mM) caused a 10-fold rightward shift in the concentration response curve of cytokines. These results do not appear to support the ultimate role of oxidant stress in NF-κB activation in these cells. To elucidate the role of proteolysis in NF-κB activation, we tested the effects of distinct protease inhibitors with different activities towards the proteasome pathway. Calpain inhibitors I and II, but not leupeptin, temporarily stabilize phosphorylated I-κB and cause a strong positive immunoreactivity in the high molecular weight region of 250-100 kDa, consistent with accumulation of polyubiquitinylated I-κB. Moreover, both Calpain inhibitors, but not leupeptin attenuated p65 nuclear translocation.

Keywords

Proteasome Pathway Bacterial Lipopolysaccharide Rightward Shift Calpain Inhibitor Concentration Response Curve 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Nandor Marczin
    • 1
  • Ginette Hoare
    • 1
  • Karen Morrison
    • 1
  • Adrian Chester
    • 1
  • Magdi Yacoub
    • 1
  1. 1.Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Technology, and MedicineHarefield HospitalUK

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