Mechanisms of HSF1-mediated Repression of TNF-alpha

HSF1 介导的 TNF-α 抑制机制

• 批准号: 6875575
• 负责人: JEFFREY D HASDAY
• 金额: $ 30.35万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6875575
• 关键词: animal tissue; biological signal transduction; chromatin; cytokine; gene expression; gene induction /repression; genetic promoter element; genetic regulation; heat shock proteins; hyperthermia; leukemia inhibitory factor; lipopolysaccharides; macrophage; phosphorylation; tissue /cell culture; toll like receptor; transcription factor; tumor necrosis factor alpha

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.

描述（由申请人提供）：发烧是一种进化上保守的反应，其关键特征是核心温度的暂时、有调节的升高。我们已经证明，温度升高本身就是一种免疫调节剂，其一个关键特征是减弱促炎细胞因子、肿瘤坏死因子-α (TNF)。我们发现，亚热休克发热范围过高 (FRH) 会激活热/应激激活的转录因子热休克因子 1 (HSF1)，形成一种独特的阻遏物形式。我们在鼠 TNFα 5'UTR 中鉴定了一个 HSF1 结合位点，该位点抑制 TNF 启动子活性，并在 TNF 启动子中发现了其他 HSF1 依赖性阻遏物的证据。在暴露于 FRH 的巨噬细胞中，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：在反式阻遏蛋白存在的情况下，TNF 转录是如何开始的？在这方面，我们发现HSF1在LPS处理后短暂失活，HSF1失活与TNF转录的开始一致，并且随后的HSF1重新激活与TNF转录沉默一致。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 60 分钟周期内的去磷酸化引起的。我们假设 LPS 激活 HSF1 磷酸化/去磷酸化循环，并且该过程暂时限制 TNF 表达。本研究计划的总体目标是阐明 HSF1 改变巨噬细胞中 TNF 表达的分子机制。具体来说，我们将使用新的HSF1无效巨噬细胞模型来：（i）定义HSF1抑制TNF表达的机制，重点关注其与近端TNF启动子和相关增强体的相互作用，及其对局部染色质结构的影响； (ii) 鉴定 TNF 反式阻抑所需的 HSF1 结构域； (iii) 阐明 LPS 激活的磷酸化事件短暂失活 HSF1 反式抑制，从而允许 FRH 处理的细胞中短暂表达 TNF； (iv)确定HSF1是否直接阻断TLR信号传导、HSF1是否直接结合至受抑制的信号传导元件，并鉴定活性HSF1结构域。拟议的研究将为调节宿主防御的生化事件如何因发烧期间发生的温度升高而改变提供新的见解。