Propanil inhibits tumor necrosis factor-alpha production by reducing nuclear levels of the transcription factor nuclear factor-kappab in the macrophage cell line ic-21.
Tumor necrosis factor-alpha (TNF-alpha) is an essential proinflammatory cytokine whose production is normally stimulated by bacterial cell wall components, such as lipopolysaccharide (LPS), during an infection. Macrophages stimulated with LPS in vitro produce several cytokines, including TNF-alpha. LPS-stimulated primary mouse macrophages produced less TNF-alpha protein and message after treatment with the herbicide propanil (Xie et al., Toxicol. Appl. Pharmacol. 145, 184-191, 1997). Nuclear factor-kappaB (NF-kappaB) tightly regulates TNF-alpha transcription. Therefore, as a step toward understanding the mechanism of the effect of propanil on TNF-alpha transcription, IC-21 cells were transfected with a TNF-alpha promoter-luciferase construct, and the effect of propanil on luciferase activity was measured. Cells transfected with promoter constructs containing a kappaB site showed decreased luciferase activity relative to controls after propanil treatment. These observations implicated NF-kappaB binding as an intracellular target of propanil. Further studies demonstrated a marked reduction in the nuclear levels of the stimulatory p65 subunit of NF-kappaB after propanil treatment, as measured by fluorescence confocal microscopy and Western blot analysis. The p50 subunit of NF-kappaB was not found to be reduced after propanil exposure by Western blot. Electrophoretic mobility gel shift assays showed decreased DNA binding of both p65/p50 heterodimers and p50/p50 homodimers to the kappaB3 site of the TNF-alpha promoter of propanil-treated cells. The marked reduction in nuclear p65/p50 NF-kappaB levels and diminished binding to the TNF-alpha promoter in propanil-treated cells are consistent with reduced TNF-alpha levels induced by LPS.
Frost
L L
LL
Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia, 26506, USA.
Neeley
Y X
YX
Schafer
R
R
Gibson
L F
LF
Barnett
J B
JB
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004586:Electrophoresis; D006540:Herbicides / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011411:Propanil / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D006540:Herbicides; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D011411:Propanil; D004247:DNA; D008156:Luciferases