Bacterial endotoxin modifies heat shock factor-1 activity in RAW 264.7 cells: implications for TNF-alpha regulation during exposure to febrile range temperatures.
Recent studies have identified heat shock factor (HSF)-1, the predominant heat/stress-stimulated transcriptional activator of heat shock protein genes as a repressor of certain cytokine genes, including TNF-alpha and IL-1beta. We previously showed that exposing macrophages to febrile-range temperature (FRT; 39.5 degrees C) activates HSF-1 to a DNA binding form that does not activate heat shock protein gene transcription, but apparently represses TNF-alpha and IL-1beta transcription. Prewarming macrophages to 39.5 degrees C for 30 min prior to stimulation with bacterial lipopolysaccharide (LPS) does not change the induction of TNF-alpha transcription, but markedly reduces its duration. This raised the question of how TNF-alpha transcription could occur at all in the presence of activated HSF-1. We used RAW 264.7 cells to test the hypothesis that macrophage activation triggers a transient reversal of HSF-1-mediated repression, thereby allowing induction of TNF-alpha transcription. Electrophoretic mobility shift assays revealed that LPS triggers a transient inactivation of HSF-1 that temporally correlates with TNF-alpha transcription and was associated with a transient increase in HSF-1 molecular weight, a decrease in its pI, and appearance of HSF-1 phosphorylating activity. The serine/threonine phosphatase inhibitor, calyculin A, blocked the inhibitory affect of FRT on LPS-induced TNF-alpha generation and prevented the re-activation of HSF-1. We propose that LPS stimulation of FRT-exposed macrophages stimulates a sequential phosphorylation and dephosphorylation of HSF-1, causing a cycle of inactivation and reactivation of HSF-1 repressor activity that allows a temporally-limited period of gene transcription.
Singh
Ishwar S
IS
Department of Medicine, University of Maryland and Medical and Research Services of the Baltimore VA Medical Center, Baltimore, Maryland 21201, USA.
He
Ju-Ren
JR
Hester
Lisa
L
Fenton
Matthew J
MJ
Hasday
Jeffrey D
JD
D000818:Animals; D018929:Cell Culture Techniques; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology*; D005334:Fever; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D007375:Interleukin-1 / Q000096:biosynthesis / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology* / Q000633:toxicity*; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology; D051379:Mice; D010766:Phosphorylation; D014157:Transcription Factors; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000494:pharmacology
D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha