Mechanisms of HSF1-mediated Repression of TNF-alpha

描述（由申请人提供）：发热是一种进化上保守的反应，其关键特征是核心温度的暂时性、调节性升高。我们已经表明，温度升高本身是一种免疫调节剂，其中一个关键特征是促炎细胞因子肿瘤坏死因子-α（TNF）的衰减。我们发现，亚热休克发热范围高温（FRH）激活热/应激激活转录因子，热休克因子-1（HSF 1）的一个独特的阻遏形式。我们在鼠TNF α 5 'UTR中鉴定了抑制TNF启动子活性的HSF 1结合位点，并发现了TNF启动子中存在其他HSF 1依赖性抑制物的证据。在暴露于FRH的小鼠中，TNF转录在用细菌脂多糖（LPS）处理后正常开始，但提前结束，从而将TNF表达降低至短暂的短脉冲。这就提出了一个问题，即在存在反式阻遏物的情况下，TNF转录是如何开始的。在这方面，我们发现，HSF 1是短暂的灭活LPS处理后，HSF 1失活与TNF转录的开始，和随后的HSF 1再激活与TNF转录沉默。初步数据表明，HSF 1的失活和再活化是由其磷酸化和随后的去磷酸化在60分钟的周期。我们假设LPS激活了HSF 1磷酸化/去磷酸化的循环，并且该过程暂时限制了TNF的表达。本研究的总体目标是阐明HSF 1修饰巨噬细胞中TNF表达的分子机制。具体地说，我们将使用新的HSF 1-null巨噬细胞模型：（i）确定HSF 1抑制TNF表达的机制，重点是它与近端TNF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）鉴定TNF反式抑制所需的HSF 1结构域;（iii）阐明瞬时抑制HSF 1反式阻遏的LPS激活的磷酸化事件，从而允许在FRH处理的细胞中瞬时TNF表达;和（iv）确定HSF 1是否直接阻断TLR信号传导，HSF 1是否直接结合至受抑制的信号传导元件，并鉴定活性HSF 1结构域。拟议中的研究将提供新的见解，以了解调节宿主防御的生化事件如何被发烧期间发生的温度升高所改变。

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.