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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: These results suggest that the activation of NF-KappaB mediates IL-1beta induced ICAM-1 expression in A549 cells.

Reference gene selection for real-time polymerase chain reaction in human lung cells subjected to cyclic mechanical strain.

BACKGROUND AND OBJECTIVE: The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. METHODS: The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. RESULTS: The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. CONCLUSIONS: These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific.