Fever is a complex systemic response to infection and injury whereas heat shock (HS) is a phylogenetically ancient, highly conserved cellular response to stress. Although fever and HS are two distinct responses, they appear to be partially overlapping processes that share certain common signaling pathways. One common phenomenon between fever and HS is the activation of heat shock factor-1 (HSF1), a stress-activated transcription factor that is essential for generating the HS response. HSF1 was originally identified as a transcriptional activator of heat shock protein genes but recent studies, including ours, suggest that it can also act as a repressor of certain proinflammatory cytokine and early response genes. Our long-term goal is to understand the role of HSF1 in regulating immune responses during fever. In the present proposal we will elucidate the signaling events that are activated at febrile range temperature (FRT) and determine how these events mediate post-translational processing of HSFland regulate its transactivation and repressor function at FRT. Using the RAW 264.7 murine macrophage cell line as a model and 39.5 degrees C as the febrile range incubation temperature we will elucidate the kinase(s) that are activated and identify the sites that are phosphorylated by each in HSFI. We will then determine the functional consequence of each phosphorylation event in terms of HSF1 DNA-binding and repressor activity of HSF1. Using co-immunoprecipitation, GST-pull down and mass spectrometric procedures we will identify the proteins that associate with HSF1 at FRT and determine how the phosphorylation events that occur at FRT modify these interactions. Our previous studies support a central role for HSF1 as the central mediator of FRT-induced tumor necrosis factor alpha gene repression. The proposed research will help us understand how the heat shock-activated transcription factor, HSF1, is modified at FRT to generate a unique repressor form and how the signaling cascades that are activated at FRT regulate HSF1 function. Moreover, these studies may lead to new concepts about fever management and using the heat shock/fever pathway to regulate expression of inflammatory mediators.
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