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，其主要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是一种免疫调节剂，其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，亚热休克热范围热休克（FRH）激活热/应激激活转录因子，热休克因子-1 （HSF1）到一个独特的抑制形式。我们在小鼠TNFalpha 5'UTR中发现了抑制TNF启动子活性的HSF1结合位点，并在TNF启动子中发现了其他HSF1依赖性抑制因子的证据。在暴露于FRH的噬细胞中，TNF转录在细菌脂多糖（LPS）治疗后正常开始，但提前结束，从而使TNF表达降低到短暂的短脉冲。这就提出了一个问题，TNF转录是如何在反式抑制因子存在的情况下开始的。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，随后HSF1再激活与TNF转录沉默一致。初步数据表明HSF1的失活和再激活是由其磷酸化和随后的去磷酸化在60分钟的周期内引起的。我们假设LPS激活了HSF1磷酸化/去磷酸化的循环，这一过程暂时限制了TNF的表达。本研究计划的总体目标是阐明HSF1修饰巨噬细胞中TNF表达的分子机制。具体而言，我们将使用新的HSFl-null巨噬细胞模型：(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，以及其对局部染色质结构的影响；（ii）确定TNF转抑制所需的HSF1结构域；（iii）阐明lps激活的磷酸化事件(s)，使HSF1反式抑制瞬间失活，从而允许frh处理的细胞中瞬时表达TNF；（iv）确定HSF1是否直接阻断TLR信号，HSF1是否直接与被抑制的信号元件结合，并识别活跃的HSF1结构域。提出的研究将提供新的见解，调节宿主防御的生化事件是如何被发烧期间发生的温度升高所改变的。