Effect of activation of nuclear factor-KappaB on modulation of secretion of intercellular adhesion molecule-1 by A549 cell

Abstract

To investigate the effects of interleukin-1beta (IL-1beta) on intercellular adhesion molecule-1 (ICAM-1) expression on A549 cell and underlying signal transduction pathways. A549 cells were pre-incubated with SC-514 [nuclear factor-KappaB (NF-KappaB) inhibitor (IKappaB) kinase-2 (IKK-2) inhibitor] and/or pre-treated with 1 microg/L IL-1beta. The phosphorylated IKappaBalpha (pIKappaBalpha) and degradation of IKappaBalpha were determined by Western blotting with specific antibody at 5, 10, 30 and 60 minutes. Laser scanning confocal microscope (LSCM) was used to examine the nuclear translocation of p65 at 30 minutes after stimulation. The DNA binding activity of p65 in nuclear extracts was detected at 1 hour following IL-1beta treatment. Chromatin immunoprecipitation (ChIP) assays combined with polymerase chain reaction (PCR) were used to evaluate interaction between p65 and ICAM-1 promoter site DNA at 1 hour after stimulation. The expression of ICAM-1 mRNA was assessed by reverse transcription (RT)-PCR at 4 hours, and the ICAM-1 expression on A549 cell surface was measured by enzyme linked immunosorbent assay (ELISA) at 24 hours after IL-1beta was added. IL-1beta induced rapid pIKappaBalpha augmentation and its subsequent degradation. LSCM graphs showed that IL-1beta stimulated the translocation of p65 from the cytosol to the nucleus. IL-1beta significantly increased the DNA binding ability of p65 (P<0.01) in cell nuclear extracts. ChIP-PCR suggested that both acetylated histone 4 and p65 were recruited to ICAM-1 promoter. IL-1beta significantly augmented ICAM-1 mRNA level at 4 hours and expression of ICAM-1 on A549 cell surface at 24 hours (both P<0.01). The IKK-2 inhibitor, SC-514, inhibited IL-1beta induced IKappaBalpha protein activity, blocked p65 nuclear translocation, caused a significant reduction in IL-1beta induced DNA binding activity for p65 and ICAM-1 mRNA expression, and suppressed ICAM-1 expression on A549 cell surface (all P<0.01). These results suggest that the activation of NF-KappaB mediates IL-1beta induced ICAM-1 expression in A549 cells.