請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64470
標題: | 探討zVAD誘導TLR所活化的巨噬細胞壞死及自體吞噬死亡的作用機制 Molecular mechanisms of zVAD-induced necroptosis and autophagic cell death in TLR-stimulated macrophages |
作者: | Wei-Chu Chuang 莊惟筑 |
指導教授: | 林琬琬 |
關鍵字: | 巨噬細胞,細胞凋亡,自體吞噬死亡,zVAD,TLR, macrophage,necroptosis,autophagic cell death,zVAD,TLR, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 細胞凋亡主要是由蛋白分解酵素 caspase 來執行。然而有報導指出,細胞凋亡酵素 caspase 也參與其他形式的死亡,其中包含細胞壞死及自體吞噬死亡。Necroptosis 是一種計畫性的細胞壞死,常在死亡受體或 TLR 被啟動且 caspases被抑制(例如在 pan-caspase peptide inhibitor,zVAD 存在下)時會被引發。已知
caspase 的下游受質 RIP1 kinase,對於粒線體產生氧化自由基(ROS)及 necroptosis扮演重要的角色。巨噬細胞的死亡在生理及病理角色扮演許多不同的影響及重要性。有報導指出,在 lipopolysaccharide(LPS)和 zVAD 共同存在下,會使巨噬細胞走向計畫性的細胞壞死及自體吞噬死亡。在這篇研究,我們有興趣了解細胞壞死的訊息傳遞及分子作用機制的過程。首先,我們確實在 LPS(TLR4 ligand)或 polyIC(TLR3 ligand)與 zVAD 共同處理骨髓分化的巨噬細胞後,會產生細胞 的 壞 死 ; 這 主 要 是 因 為 測 得 LDH 的 釋 放 及 發 現 RIP1 kinase 抑 制 劑 (necrostatin-1,Nec-1 )可以保護細胞避免死亡。另一方面,在電子顯微鏡觀察到自噬小體形成及 LC3-II 蛋白的表現增加,得證細胞自體吞噬的存在。此外,我們發現 zVAD 不影響 LPS 或 polyIC 活化 IKK、JNK、p38 的作用,然而會增加IRF3 和 STAT1 的活性,及 type I IFN 基因和蛋白的表現,且 zVAD 可以抑制 LPS或 polyIC 引起的 ERK 及 Akt 磷酸化。其中上述被 zVAD 增加的作用可被 Nec-1抑制,而 ERK、Akt 的抑制作用卻不受 Nec-1 影響。我們的結果也顯示 TLR ligand和 zVAD 所導致的細胞死亡有氧化自由基及 type I IFNs 的參與。我們發現 ROS 抑制劑 (BHA)、IFN 受體的中和抗體 (IFNR-Ab)、JAK 抑制劑 (AZD1480) 可以保護細胞的活性。另外,LPS+zVAD 促使的 ROS 可被 Nec-1、IFNR-Ab 及 AZD1480 所抑制,且此細胞死亡及 ROS 的產生皆不在 STAT1 基因移除的巨噬細胞發現。最後,我們也發現 RIP1,STAT1 及 ROS 參與在 LPS+zVAD 所表現的 LC3-II,然而 ERK 及 PI3K 的抑制作用並不參與在其中。綜合以上結果,我們在 TLR 誘發巨噬細胞產生 necroptosis 型的細胞壞死過程中,揭露一個新的分子調控路徑: 即在 caspase 抑制情況下,TLR 會藉由加強活化 TRIF-RIP1-IRF3 的訊息傳遞路徑而增加 IFNs-IFNR-JAK-STAT1 的自泌素的調節作用,進而導致 ROS 產生及細胞自體吞噬死亡。 Apoptotic caspases are proteolytic enzymes mainly inducing apoptosis. It has been reported that many apoptotic caspases also participate in other signaling pathways that promote other types of cell death including necroptosis and autophagic cell death. Necroptosis is a type of programmed necrosis, induced by death receptor or TLR ligands under condition of caspase inhibition (e.g. in the presence of zVAD, a pan-caspase peptide inhibitor). Studies have shown RIP1 kinase, a substrate of caspases, as a key player for inducing mitochondria-derived ROS production and consequently the necroptosis. Macrophage cell death plays a role in many physiological and pathological conditions. There are reports indicating that programmed necroptosis and autophagic cell death are involved in caspase-independent macrophage cell death induced by co-treatment with lipopolysaccharide (LPS) and zVAD. In this study, we interested to further elucidate the signaling pathways and molecular mechanisms underlying this necroptotic process. First we indeed found that primary bone marrow-derived macrophages treated with LPS (TLR4 ligand)+zVAD or polyIC (TLR3 ligand)+zVAD display cell death with necrotic and autophagic features. The former is evidenced by LDH release and the prevention of cell death by necrostatin-1 (a specific RIP1 kinase inhibitor), while the latter is evidenced by electron microscopic observation of autophagosome and biochemically increased LC3-II expression. Moreover, although zVAD did not affect LPS- or polyIC-induced IKK, JNK and p38 activation, it enhanced IRF3 and STAT1 activation as well as type I IFN gene and protein expression. Of note, zVAD also inhibited LPS- or polyIC-induced ERK and Akt phosphorylation. Interestingly the above enhancing actions of zVAD were inhibited by necrostatin-1, while the inhibitory actions on ERK and Akt were independent of RIP1 activity. Our data further support the involvement of autocrine IFNs action in ROS-dependent necroptosis, as cell death induced by TLR ligands with zVAD co-treatment was prevented by ROS scavenger BHA, neutralizing antibody targeting IFNR and JAK inhibitor AZD1480. Consistently, LPS+zVAD-elicited ROS production was inhibited by necrostatin-1, IFNR antibody and AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands+zVAD were abrogated in STAT1 knockout macrophages. Finally, we showed the contribution of RIP1, STAT1 and ROS in LPS+zVAD-induced LC3-II expression, while both ERK and PI3K inhibition are not involved. Taken together, we propose a new molecular basis for TLR-mediated necroptosis under caspase inhibition in macrophages, which involves the enhanced TRIF-RIP1-IRF3 signaling pathway contributing to autocrine action via IFNs-IFNR-JAK-STAT1, and the consequent ROS production leading to autophagic cell death. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64470 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-101-1.pdf 目前未授權公開取用 | 3.06 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。