Inhibition of tumor necrosis factor-alpha transcription in macrophages exposed to febrile range temperature. A possible role for heat shock factor-1 as a negative transcriptional regulator.
We previously reported that expression of tumor necrosis factor-alpha (TNFalpha) was attenuated in macrophages exposed to febrile range temperatures. In this study, we analyzed the influence of temperature on TNFalpha transcription in the Raw 264.7 macrophage cell line during incubation at 37 and 39.5 degrees C. The initial activation of TNFalpha transcription in response to endotoxin (LPS) was comparable in the 37 and 39.5 degrees C cell cultures, peaking within 10 min of LPS stimulation. However, the duration of transcriptional activation was markedly reduced in the 39.5 degrees C cells (30-60 min) compared with the 37 degrees C cells (2-4 h). Deletion mapping of the TNFalpha gene revealed that the proximal 85-nucleotide promoter sequence and the 5'-untranslated region were sufficient for temperature sensitivity. This sequence contains six heat shock response element (HRE) half-sites but no complete HREs. Electrophoretic mobility shift and immunoblot assays demonstrated that nuclear transclocation of heat shock factor (HSF) and its activation to a DNA-binding form occurred in the 39.5 degrees C cells in the absence of heat shock protein-70 gene activation. The proximal TNFalpha promoter/5'-untranslated region sequence competed for HSF binding to a classic HRE. Overexpression of HSF-1 reduced activity of the TNFalpha promoter. These data suggest that partial activation of HSF-1 during exposure to febrile, sub-heat shock temperatures may block TNFalpha transcription by binding to its proximal promoter or 5'-untranslated region.
Singh
I S
IS
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Maryland School of Medicine, the University of Maryland at Baltimore Cytokine Core Laboratory, Maryland 21201, USA.
Viscardi
R M
RM
Kalvakolanu
I
I
Calderwood
S
S
Hasday
J D
JD
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology*; D005334:Fever / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology*; D000076249:Heat Shock Transcription Factors; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000502:physiology; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha