Chen Huaqun, Wu Yifan, Zhang Yiqing, Jin Lina, Luo Lan, Xue Bin, Lu Chen, Zhang Xiran, Yin Zhimin
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 122 Ninghai Road, Nanjing 210097, Jiangsu Province, PR China.
FEBS Lett. 2006 May 29;580(13):3145-52. doi: 10.1016/j.febslet.2006.04.066. Epub 2006 May 2.
Inducible heat shock protein 70 (Hsp70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological and pathological processes and is essential for activating NF-kappaB signaling pathway in response to bacterial lipopolysaccharide (LPS). Here we report a novel mechanism of Hsp70 for preventing LPS-induced NF-kappaB activation in RAW264.7 macrophage-like cells. Our results show that Hsp70 can associate with TRAF6 physically in the TRAF-C domain and prevent TRAF6 ubiquitination. The stimulation of LPS dissociates the binding of Hsp70 and TRAF6 in a time-dependent manner. Hsp70 inhibits LPS-induced NF-kappaB signaling cascade activation in heat-shock treated as well as Hsp70 stable transfected RAW264.7 cells and subsequently decreases iNOS and COX-2 expression. Two Hsp70 mutants, Hsp70DeltaC(1-428aa) with N-terminal ATPase domain and Hsp70C(428-642aa) with C-terminal domain, lack the ability to influence TRAF6 ubiquitination and TRAF6-triggered NF-kappaB activation. Taken together, these findings indicate that Hsp70 inhibits LPS-induced NF-kappaB activation by binding TRAF6 and preventing its ubiquitination, and results in inhibition of inflammatory mediator production, which provides a new insight for analyzing the effects of Hsp70 on LPS-triggered inflammatory signal transduction pathways.
诱导型热休克蛋白70（Hsp70）是在正常细胞生长以及病理生理条件下维持细胞完整性的最重要的热休克蛋白之一。肿瘤坏死因子受体相关因子6（TRAF6）是一种关键的信号转导分子，可调节多种生理和病理过程，并且对于响应细菌脂多糖（LPS）激活核因子κB信号通路至关重要。在此，我们报告了Hsp70在RAW264.7巨噬细胞样细胞中预防LPS诱导的核因子κB激活的新机制。我们的结果表明，Hsp70可在TRAF-C结构域与TRAF6发生物理结合，并阻止TRAF6泛素化。LPS刺激以时间依赖性方式使Hsp70与TRAF6的结合解离。Hsp70在热休克处理的以及Hsp70稳定转染的RAW264.7细胞中抑制LPS诱导的核因子κB信号级联激活，随后降低诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的表达。两个Hsp70突变体，具有N端ATP酶结构域的Hsp70DeltaC（1-428aa）和具有C端结构域的Hsp70C（428-642aa），缺乏影响TRAF6泛素化和TRAF6触发的核因子κB激活的能力。综上所述，这些发现表明Hsp70通过结合TRAF6并阻止其泛素化来抑制LPS诱导的核因子κB激活，并导致炎症介质产生受到抑制，这为分析Hsp70对LPS触发的炎症信号转导通路的影响提供了新的见解。
