Mechanisms of HSF1-mediated Repression of TNF-alpha

描述(申请人提供)：发热是一种进化上保守的反应，其关键特征是核心体温的暂时性、可调节的升高。我们已经证明，温度升高本身是一种免疫调节剂，其关键特征之一是促炎症细胞因子肿瘤坏死因子-α(TNF)的减弱。我们发现，亚热休克热疗(FRH)激活了热/应激激活的转录因子热休克因子-1(HSF1)，使其以一种独特的抑制形式存在。我们在小鼠肿瘤坏死因子α5‘非编码区中发现了一个抑制肿瘤坏死因子启动子活性的HSF1结合位点，并在肿瘤坏死因子启动子中发现了其他依赖于HSF1的抑制物的证据。在暴露于FRH的巨噬细胞中，经细菌脂多糖(LPS)处理后，肿瘤坏死因子的转录正常开始，但提前结束，从而将肿瘤坏死因子的表达减少到短暂的短脉冲。这就提出了一个问题，即在存在反式抑制子的情况下，肿瘤坏死因子的转录是如何开始的。在这一点上，我们发现HSF1在脂多糖处理后被瞬时失活，HSF1失活与肿瘤坏死因子转录开始相一致，随后的HSF1重新激活与肿瘤坏死因子转录沉默相一致。初步数据表明，HSF1的失活和重新激活是由其磷酸化和随后的去磷酸化在60分钟的循环中引起的。我们假设内毒素激活了HSF1磷酸化/去磷酸化的循环，这一过程暂时限制了肿瘤坏死因子的表达。这项研究计划的总体目标是阐明HSF1改变巨噬细胞中肿瘤坏死因子表达的分子机制。具体地说，我们将使用新的hsfl缺失的巨噬细胞模型来：(I)确定hsf1抑制肿瘤坏死因子表达的机制，重点是它与邻近的肿瘤坏死因子启动子和相关的增强体的相互作用，以及它对局部染色质结构的影响；(Ii)确定hsf1转录抑制所需的hsf1结构域；(Iii)阐明脂多糖激活的磷酸化事件(S)，它可以瞬时失活hsf1的反式抑制，从而允许在FRH处理的细胞中瞬时表达肿瘤坏死因子；以及(Iv)确定hsf1是否直接阻断TLR信号，是否直接与被抑制的信号元件结合，并确定活性的hsf1结构域。拟议中的研究将为调节宿主防御的生化事件如何被发烧期间发生的温度上升所改变提供新的见解。

DESCRIPTION (provided by applicant): Fever is an evolutionarily conserved response, the key feature of which is a temporary, regulated increase in core temperature. We have shown that the temperature increase itself is an immune modulator, one key feature of which is attenuation of the proinflammatory cytokine, tumor necrosis factor-alpha(TNF). We showed that sub-heat shock febrile-range hyperthermia (FRH) activates the heat/stress-activated transcription factor, heat shock factor-1 (HSF1) to a distinct repressor form. We identified an HSF1 binding site in the murine TNFalpha 5'UTR that represses TNF promoter activity and found evidence of additional HSF1-dependent repressors in the TNF promoter. In rnacrophages exposed to FRH, TNF transcription begins normally after treatment with bacterial lipopolysaccharide (LPS), but ends early, thereby reducing TNF expression to a brief short pulse. This raised the question of how TNF transcription could begin at all in the presence of a trans-repressor. In this regard, we found that HSF1 is transiently inactivated after LPS treatment that HSF1 inactivation coincides with onset of TNF transcription, and that subsequent HSF1 reactivation coincides with TNF transcriptional silencing. Preliminary data indicates that the inactivation and reactivation of HSF1 is caused by its phosphorylation and subsequent dephosphorylation over a 60-minute cycle. We hypothesize that LPS activate a cycle of HSF1 phosphorylation/dephosphorylation and that this process temporally restricts TNF expression. The overall goal of this research proposal is to elucidate the molecular mechanisms through which HSF1 modifies TNF expression in macrophages. Specifically, we will use novel HSFl-null macrophage model to: (i) define the mechanisms through which HSF1 represses TNF expression focusing on its interaction with the proximal TNF promoter and the associated enhanceosome, and its effect on local chromatin structure; (ii) identify HSF1 domains required for transrepression of TNF; (iii) elucidate the LPS-activated phosphorylation event(s) that transiently inactivate HSF1 trans-repression, thus allowing transient TNF expression in FRH-treated cells; and (iv) determine whether HSF1 directly blocks TLR signaling, whether HSF1 binds directly to the inhibited signaling elements, and identify the active HSF1 domains. The proposed studies will provide new insight into how the biochemical events that regulate host defenses is modified by the temperature increase that occurs during fever.