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.