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Bacterial endotoxin modifies heat shock factor-1 activity in RAW 264.7 cells: implications for TNF- regulation during exposure to febrile range temperatures

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Maryland and Medical and Research Services of the Baltimore VA Medical Center, Baltimore, Maryland, USA

Ju-Ren He

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Maryland and Medical and Research Services of the Baltimore VA Medical Center, Baltimore, Maryland, USA

Lisa Hester

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Maryland and Medical and Research Services of the Baltimore VA Medical Center, Baltimore, Maryland, USA

Matthew J. Fenton

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Maryland and Medical and Research Services of the Baltimore VA Medical Center, Baltimore, Maryland, USA

Jeffrey D. Hasday

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Maryland and Medical and Research Services of the Baltimore VA Medical Center, Baltimore, Maryland, USA, jhasday{at}umaryland.edu

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- and IL-1ß. We previously showed that exposing macrophages to febrile-range temperature (FRT; 39.5°C) activates HSF-1 to a DNA binding form that does not activate heat shock protein gene transcription, but apparently represses TNF- and IL-1ß transcription. Prewarming macrophages to 39.5°C for 30 min prior to stimulation with bacterial lipopolysaccharide (LPS) does not change the induction of TNF- transcription, but markedly reduces its duration. This raised the question of how TNF- 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- transcription. Electrophoretic mobility shift assays revealed that LPS triggers a transient inactivation of HSF-1 that temporally correlates with TNF- 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- 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 re-activation of HSF-1 repressor activity that allows a temporally-limited period of gene transcription.

Key Words: Endotoxin • heat shock factor-1 • RAW 264.7 cells • TNF- regulation • febrile range temperatures

Journal of Endotoxin Research, Vol. 10, No. 3, 175-184 (2004)
DOI: 10.1177/09680519040100030401


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