Febrile range temperature represses TNF-alpha gene expression in LPS-stimulated macrophages by selectively blocking recruitment of Sp1 to the TNF-alpha promoter.
We have previously shown that exposure to febrile-range temperature (FRT, 39.5 degrees C) reduces LPS-induced TNF-alpha transcription in mouse macrophages through at least two mechanisms: (1) by directly recruiting heat shock factor-1 (HSF-1) to a heat shock response element present in the TNF-alpha 5'-UTR and (2) by markedly reducing LPS-induced recruitment of NFkappaB-p65 to the kappaB enhancer (at -510) in the TNF-alpha gene. In the present study, we used EMSA and chromatin immunoprecipitation assays to further analyze the complex effects of FRT on the recruitment of transcription factors and co-activators on the TNF-alpha gene in LPS-stimulated RAW 264.7 mouse macrophages. Our results showed that in FRT-exposed RAW cells, HSF-1 was recruited only to the 5'-UTR site, and no additional interaction was evident in the TNF-alpha gene up to 1,300 nt upstream of the transcription start site. Similarly, FRT exposure selectively reduced LPS-induced NFkappaB-p65 recruitment to the kappaB enhancer site at -510 without affecting the other three kappaB enhancer sites present in the TNF-alpha 5'-flanking sequence. Finally, we found that FRT exposure abrogated LPS-stimulated recruitment of Sp1 to the proximal TNF-alpha promoter without any change in associated histone H3 acetylation around the TNF-alpha promoter and despite a marked increase in the total intra-nuclear Sp1 DNA binding activity. In conclusion, our studies further emphasize the complex and redundant control of TNF-alpha transcription and identify additional potential mechanisms through which FRT exposure may reduce TNF-alpha expression by selectively modifying gene-specific recruitment of transcription factors to the proximal TNF-alpha promoter.
Cooper
Zachary A
ZA
Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Singh
Ishwar S
IS
Hasday
Jeffrey D
JD
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D020121:5' Untranslated Regions; D008070:Lipopolysaccharides; D016329:Sp1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha