HSP27通过其自身磷酸化促进TLR4下调进而负反馈调控LPS促炎信号转导的机制研究

TLR4 is a receptor in the cell membrane which mediats innate immunity and inflammation and can be activated by LPS. HSP27(heat shock protein 27) is a kind of protein chaperon in cells and can be phosphorylated by TLR4 downstream kinase MK2. However, the relationship between HSP27 phosphorylation and TLR4 signaling is still unclear. Our recent study showed that Phosphorylated HSP27 promoted membrane TLR4 decrease in macrophages after LPS stimulation. TLR4 mediated the release of pro-inflammation factors, such as HMGB1, was enhanced if HSP27 phosphorylation was inhibited. LPS stimulation increased the binding between HSP27 and TLR4, and HSP27 phosphorylation was essential to this binding. These results suggested that HSP27 phosphorylation might provide a negative feedback regulatory mechanism in TLR4 signaling during LPS stimulation. In the present research project, we will investigate the role of HSP27 phosphorylation in maintaining the balance between TLR4 activation and degradation in LPS stimulated macrophages. The study includes 1) HSP27 phosphorylation under LPS stimulation, 2)the regulatory effect of phosphorylated HSP27 on the TLR4 signal transduction from plasma mambrane including the release of proinflammatory factors, 3) the molecular mechanism by which phosphorylated HSP27 regulated TLR4 endocytosis and degradation, 4) the anti-inflammatory effects of the HSP27 phosphorylation in animal endotoxic shock and sepsis models. These investigation will uncover the negative feedback regulation of LPS-TLR4 inflammatory signaling by HSP27 phosphorylation. The results from this research project will provide a new insight for realizing the novel function of HSP27 phosphorylation, and will help us to find the new strategy for anti-inflammation therapy.

TLR4是固有免疫和炎症反应的相关受体，TLR4信号通路过度激活是引起重度炎症疾病的重要原因。单核巨噬细胞TLR4通路的调控是影响炎症反应的关键，但尚不清楚是否存在负反馈调控TLR4的机制。HSP27可被TLR4下游激酶磷酸化，我们近期的研究表明，LPS刺激后磷酸化的HSP27与TLR4结合；HSP27磷酸化促进TLR4从细胞膜上减少，抑制HSP27磷酸化增加TLR4介导的促炎因子释放。提示LPS通过TLR4引起的HSP27磷酸化可负反馈调控TLR4。本项目拟研究：1、HSP27磷酸化抑制TLR4炎症信号转导，特别是抑制死亡相关的晚期炎症因子HMGB1释放的作用；2、HSP27磷酸化促进TLR4被内吞和降解的机制；3、HSP27磷酸化对小鼠脓毒症和内毒素休克模型死亡率的影响。拟阐明TLR4激活引起的HSP27磷酸化对炎症信号通路的负反馈调控机制，为控制过度炎症反应、治疗脓毒症提供依据。

Hsp27是小热休克蛋白家族重要成员之一，其生理功能主要包括保护细胞免受各种应激因素如自由基、高温、缺血和毒性物质的损伤，促进蛋白质的正确折叠、组装，参与细胞的发育，增殖、分化及细胞凋亡的信号调节和肌动蛋白丝重建等。我们发现， LPS刺激引起Hsp27发生磷酸化修饰。在LPS刺激的情况下，抑制Hsp27磷酸化，以 NF- κB 和 IRF3 的激活分别为标志的 TLR4 下游 MyD88 依赖型和 MyD88 非依赖型 /TRIF 依赖型两条通路均被下调。通过免疫共沉淀、激光共聚焦等分子生物学实验发现，磷酸化Hsp27通过与TLR4结合，促进TLR4的内吞和泛素化降解，下调LPS诱导的TLR4信号通路，阐明了磷酸化Hsp27参与调控LPS引起TLR4通路炎症应答的机制。.在LPS刺激后，Hsp27在丝氨酸残基Ser15/78发生磷酸化并从细胞质转移到细胞核中。相应地，LPS刺激增加了CBP水平并促使其进入细胞核。在细胞核中，磷酸化Hsp27结合CBP并抑制CBP乙酰转移酶活性和随后的CBP依赖性的HMGB1乙酰化与胞外释放。进一步证明了， Hsp27在维持细胞稳态并抑制过度炎症反应中起着重要作用。.脓毒症是一种全身性炎症综合征，可导致致命性的器官损伤，研究发现，即使在脓毒症造模后8小时开始给予CBP抑制剂SGC-CBP30治疗也会提高模型小鼠的生存率，降低血液循环中HMGB1的水平，并改善脓毒症相关器官组织学损伤。