Differential regulation of nitric oxide synthase mRNA expression by lipopolysaccharide and pro-inflammatory cytokines in fetal hepatocytes treated with cycloheximide.
The effect of cycloheximide (CHX) on the mRNA expression of the cytokine-inducible, calcium-independent nitric oxide synthase (iNOS) was investigated in fetal hepatocytes stimulated with lipopolysaccharide (LPS) or pro-inflammatory cytokines. In the presence of CHX the LPS-dependent iNOS mRNA levels were reduced, whereas the response to pro-inflammatory cytokines was enhanced. Because iNOS transcription is highly dependent on the activation of nuclear factor kappaB (NF-kappaB), this factor was evaluated by electrophoretic mobility shift assays, and a close correlation between NF-kappaB activity and iNOS mRNA levels was observed. CHX itself potentiated the degradation of the IkappaB alpha and IkappaB beta inhibitory subunits (IkappaB is inhibitory kappaB) of the NF-kappaB complex, and therefore the loss of LPS-dependent iNOS mRNA expression cannot be attributed to a blockage in the activation of NF-kappaB. These results suggest the existence of a CHX-sensitive pathway in the expression of iNOS mediated by LPS, a mechanism that is not involved in the response to pro-inflammatory cytokines.
Casado
M
M
Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.
Díaz-Guerra
M J
MJ
Boscá
L
L
Martín-Sanz
P
P
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D015971:Gene Expression Regulation, Enzymologic; D020672:I-kappa B Proteins; D066298:In Vitro Techniques; D018836:Inflammation Mediators / Q000378:metabolism / Q000494:pharmacology; D007375:Interleukin-1 / Q000378:metabolism / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000196:embryology / Q000201:enzymology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
D016207:Cytokines; D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat