Tid1和CHIP在Lipopolysaccharide刺激H9c2細胞肥大及凋亡下所扮演的角色探討

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Title:	Tid1和CHIP在Lipopolysaccharide刺激H9c2細胞肥大及凋亡下所扮演的角色探討 Roles of Tid1 and CHIP on Lipopolysaccharide stimulated H9c2 Cardiomyoblast Cells
Authors:	陳佳華;Chia-Hua Chen
Contributors:	基礎醫學研究所碩士班
Keywords:	脂多醣;熱休克蛋白70羧基端作用蛋白;熱休克蛋白70;lipopolysaccharide;CHIP;Hsp70
Date:	2013-08-14
Issue Date:	2013-10-02 11:16:21 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	脂多醣 (lipopolysaccharide, LPS)不僅參與心臟的發炎反應，也可能導致細菌性敗血症並且進一步造成心血管衰竭和個體死亡。CHIP (熱休克蛋白70羧基端作用蛋白)被定義為熱休克蛋白70和90的伴侶蛋白，並且具有E3-泛素連接?的活性，而CHIP也被證實在心臟中扮演保護的角色。由先前的文獻指出，ASK1-CHIP-HSP70複合體在抑制心肌細胞凋亡和心臟功能不全中扮演一個關鍵的角色。然而，CHIP在脂多醣誘導心肌細胞肥大與凋亡反應中所扮演的角色尚未被探討。根據免疫組織染色的實驗結果，我們發現CHIP的蛋白表現在不同階段的心肌梗塞病人組織切片中有增加的趨勢。在細胞實驗中，我們也發現脂多醣和異丙(去甲)腎上腺素刺激會增加CHIP的蛋白表現量。上述結果顯示CHIP可能參與脂多醣誘導心肌細胞損傷及心肌細胞重組之過程。在進一步的實驗中，我們發現過量表現帶有HA-CHIP重組蛋白確實可以降低脂多醣所誘導的細胞肥大與細胞凋亡蛋白(例如：NFATc3, BNP, cytochrome c和 caspase 3)的表現。並且所有抑制的現象在處理CHIP的干擾RNA後均獲得回復。我們同時發現CHIP會結合NFATc3並促進NFATc3經由泛素-蛋白?系統降解因而抑制脂多醣所誘導的細胞肥大與細胞凋亡現象；況且這樣的現象必須有熱休克蛋白70的參與。根據本篇實驗的結果，CHIP是誘發心肌細胞肥大凋亡之轉錄因子NFATc3的E3-泛素連接?並經由結合NFATc3啟動泛素-蛋白?系統引導其降解，因而抑制了脂多醣所誘導的心肌細胞肥大與凋亡現象。實驗將會持續換用更多的干擾RNA和抑制劑來證明此機轉。 It is well known that lipopolysaccharide (LPS) not only participates in cardiac inflammatory response but also result in cardiovascular collapse and death during bacterial sepsis. Moreover, carboxyl terminus of Hsc70 interacting protein (CHIP), a co-chaperone of heat shock proteins (Hsp70 and Hsp90), regulates protein folding-refolding and E3 ubiquitin ligase activity, which was identified as a cardiac protect role in cardiomyocytes. Previous reports have indicated that, ASK1-CHIP-HSP70 complex played a critical role in inhibition of cardiomyocytes apoptosis and cardiac dysfunction. However, the function of CHIP on LPS-induced cardiac hypertrophy and apoptosis remains unclear. From the IHC experiment, we found CHIP protein expression was increased in different stage of human myocardial infarction tissues. Especially, CHIP was induced by LPS and Isoproterenol (ISO) treatment. Our data suggested that CHIP may involve in LPS-induced cardiac healing and remodeling. After over-expression of HA-CHIP suppressed NFATc3, BNP, cytochrome c and caspase 3 protein levels and thus reduced LPS-induced-hypertrophy and –apoptosis of cardiomyocytes. But all the effects were further reversed by treated with esiRNA of CHIP. We further found that CHIP bind directly with NFATc3 to enhance NFATc3 proteasomal degradation. However, Hsp70 is reqired for CHIP mediated NFATc3 degradation. Our study revealed that CHIP might act as an E3 ligase of NFATc3 and mediates its proteasomal-degradation; which further inhibited LPS-induced hypertrophy and apoptosis in cardimyocytes. More studies using siRNA and inhibitors will be done to prove the above mentioned mechanisms.
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