Dependence of NF-kappaB activation and free radical generation on silica-induced TNF-alpha production in macrophages.
Tumor necrosis factor alpha (TNFalpha) plays an important role in the pathogenesis of silicosis and other chronic inflammatory lung diseases. The present study investigates the role nuclear transcription factor kappaB (NF-kappaB) and oxygen free radicals in silica-induced TNFalpha production in primary alveolar macrophages and RAW 264.7 cells. Using electrophoretic mobility shift assay (EMSA) and enzyme-linked immunoadsorbent assay (ELISA), we have demonstrated that silica can induce NF-kappaB activation and TNFalpha expression in a dose-dependent manner. Transient transfection assays with a plasmid construct containing NF-kappaB binding sites linked to a reporter gene further show that silica is able to induce the transcriptional activation of NF-kappaB-dependent gene. Inhibition of NF-kappaB activation by SN50, a specific NF-kappaB blocker, abolishes silica-induced TNFalpha production. Pretreatment of the cells with catalase (H2O2 scavenger) or deferoxamine (*OH scavenger) effectively inhibits NF-kappaB and TNFalpha activation, whereas superoxide dismutase (O2 scavenger) has an opposite effect. These results indicate that silica-mediated free radical generation and NF-kappaB activation play important roles in silica-induced TNFalpha gene expression.
Rojanasakul
Y
Y
Department of Basic Pharmaceutical Sciences, West Virginia University, Health Sciences Center, Morgantown 26506, USA.
Ye
J
J
Chen
F
F
Wang
L
L
Cheng
N
N
Castranova
V
V
Vallyathan
V
V
Shi
X
X
D000818:Animals; D001483:Base Sequence; D002374:Catalase / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D015342:DNA Probes / Q000235:genetics; D003676:Deferoxamine / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012822:Silicon Dioxide / Q000633:toxicity*; D013482:Superoxide Dismutase / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D015342:DNA Probes; D016166:Free Radical Scavengers; D005609:Free Radicals; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D012822:Silicon Dioxide; D002374:Catalase; D013482:Superoxide Dismutase; D003676:Deferoxamine