Boo A. Shin

Interleukin-1β stimulates IL-8 expression through MAP kinase and ROS signaling in human gastric carcinoma cells

Recent studies have suggested that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. In this study, the effect of IL-1β on IL-8 expression in human gastric cancer TMK-1 cells and the underlying signal transduction pathways were investigated. IL-1β induced the IL-8 expression in a time- and concentration-dependent manner. IL-1β induced the activation of extracellular signal-regulated kinases-1/2 and P38 mitogen-activated protein kinase (MAPK), but not the activation of c-jun amino-terminal kinse and Akt. Specific inhibitors of MEK-1 (PD980590) and P38 MAPK (SB203580) were found to suppress the IL-8 expression and the IL-8 promoter activity. Expression of vectors encoding a mutated-type MEK-1 and P38 MAPK resulted in decrease in the IL-8 promoter activity. IL-1β also induced the production of reactive oxygen species (ROS). N-acetyl cysteine (NAC) prevented the IL-1β-induced ROS production and IL-8 expression. In addition, exogenous H2O2 could induce the IL-8 expression. Deletional and site-directed mutagenesis studies on the IL-8 promoter revealed that activator protein-1 (AP-1) and nuclear factor (NF)-κB sites were required for the IL-1β-induced IL-8 transcription. Electrophoretic mobility shift assay confirmed that IL-1β increased the DNA-binding activity of AP-1 and NF-κB. Inhibitor (PD980590, SB203580) and ROS scavenger (NAC) studies revealed that the upstream signalings for the transcription factors AP-1 and NF-κB were MAPK and ROS, respectively. Conditioned media from the TMK-1 cells pretreated with IL-1β could remarkably stimulate the in vitro growth of HUVEC and this effect was partially abrogated by IL-8-neutralizing antibodies. The above results suggest that MAPK-AP-1 and ROS-NF-κB signaling pathways are involved in the IL-1β-induced IL-8 expression and that these paracrine signaling pathways induce endothelial cell proliferation.

IL-1β induces MMP-9 via reactive oxygen species and NF-κB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2(),7()-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.