Relative contribution of transcription and translation to the induction of tumor necrosis factor-alpha by lipopolysaccharide.
The synthesis of tumor necrosis factor-alpha has been suggested to be regulated at both the transcriptional and translational levels in response to stimulation by bacterial lipopolysaccharide, although the relative contribution of these two mechanisms has not been quantitatively evaluated. Here, using the murine monocytic cell line RAW 264.7 as a model system, we show that steady-state TNF-alpha mRNA levels increase approximately 77-fold following treatment with lipopolysaccharide for 2 h and to a maximum of 164-fold after 8 h as measured by an RNase protection assay. The TNF-alpha gene transcription rate increases approximately 5-fold following exposure to lipopolysaccharide for 2 h as measured by a nuclear run-on assay. TNF-alpha mRNA stability did not change in the presence of lipopolysaccharide. A ribosomal sedimentation assay and an RNA transfection assay revealed that the translation rate of endogenous as well as transiently transfected TNF-alpha mRNAs increases only approximately 2-3-fold after stimulation with lipopolysaccharide for 2 h. Taken together, these results suggest that the large increase in the level of secreted TNF-alpha protein in RAW 264.7 cells is due primarily to activation of TNF-alpha gene transcription.
Raabe
T
T
Laboratory of Gene Regulation, The Picower Institute for Medical Research, Manhasset, New York 11030, USA.
Bukrinsky
M
M
Currie
R A
RA
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D014176:Protein Biosynthesis / Q000187:drug effects*; D012270:Ribosomes; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D015500:Chloramphenicol O-Acetyltransferase