Repression of TNFalpha gene activation at febrile range temperature through modification of recruitment of transcriptional regulators

Abstract

We have previously shown that exposure to febrile-range temperatures (FRT, 39.5-40°C) reduces lipopolysaccaride (LPS)-induced tumor necrosis factor-α (TNFα) expression, in part through the direct interaction of heat shock factor-1 (HSF-1) with the TNFα gene promoter. However, it is not known whether exposure to FRT also modifies other proximal LPS-induced signaling events or recruitment of transcriptional regulators to the TNFα promoter. Using HSF-1-null mice, we confirmed that HSF-1 is required for FRT-induced repression of TNFα in vitro by LPS-stimulated bone marrow derived macrophages and in vivo in mice challenged intratracheally with LPS. Exposing LPS-stimulated RAW 264.7 mouse macrophages to FRT reduced TNFα expression, while increasing interleukin (IL)-1β expression despite the two genes being regulated by the same MyD88-dependent pathway. Global activation of the three LPS induced signaling intermediates that lead to cytokine gene expression, ERK and p38 MAPKs and NFκB, was not affected by exposing RAW 264.7 cells to FRT as assessed by western blot analysis of ERK and p38 phosphorylation and analysis of NFκB activation by EMSA and reporter plasmid expression assays. However, chromatin immunoprecipitation (ChIP) analysis demonstrated that exposure to FRT reduced LPS-induced recruitment of NFκB-p65 to the TNFα promoter, while increasing its recruitment to the IL-1β promoter. An additional ChIP analysis shows that LPS stimulated a 90% increase in recruitment of Sp1 to the proximal TNFα promoter at 37�C, which was completely abrogated by exposure to FRT even though FRT exposure increased intranuclear Sp1 DNA-binding as measured by EMSA. LPS also stimulated recruitment of both Elk-1 and ATF-2 to the proximal promoter, but FRT exposure had no significant effect on this process. ChIP analysis of the 1452 bp TNFα 5'flanking sequence (-1300/+152) revealed no additional heat shock response elements (HSEs) and no effect of FRT on chromatin acetylation on this sequence. These data suggest that FRT exerts its effects on cytokine gene expression in a gene specific manner through downstream effects on promoter activation, rather than through proximal receptor activation/signaling events. In conclusion, we describe new mechanisms through which TNFα expression is reduced at FRT through gene-specific reduction of NFκB and Sp1 recruitment to the TNFα promoter.