3-Methyladenine調控脂多醣在巨噬細胞活化之發炎反應的機制探討

3-Methyladenine (3-MA)是現今最廣泛應用的細胞自噬抑制劑。過去認為3-MA經由阻擾class III PI3K的作用來抑制細胞自噬，但近來發現3-MA亦可對class I PI3K產生抑制作用，進而促進細胞自噬。因此近年來針對該化合物的藥理作用的專一性提出越來越多質疑，3-MA於調控細胞自噬角色也逐漸引起了爭議。過去的研究報導發現，發炎反應和細胞自噬之間存在密切的相互影響關係，而3-MA的許多非專一作用也可能與發炎反應息息相關。因此，在這個研究中我們深入探討3-MA調控脂多醣經TLR4引發之發炎反應的作用是否與PI3K/Akt/GSK3b訊息傳遞路徑有關，並釐清是否也與3-MA調節細胞自噬所扮演的角色有相關。實驗結果發現3-MA可促進脂多醣在老鼠RAW264.7巨噬細胞所引發之發炎反應，包括NF-kB的活化、iNOS及COX2的表現及一些促發炎細胞激素如IL-1b及IL-12的生成，然而卻會抑制IFNb、IL-10和IL-6等的生成。針對TLR4的上游訊息傳遞作探討，發現3-MA會正向調控TAK1、p38、JNK與p65，卻會負調控TBK1和IRF3的活性。過去的研究指出，class I PI3K下游的Akt蛋白分子對於TLR4經MyD88和TRIF所媒介的訊息傳遞路徑扮演著雙面調控的角色。我們的實驗結果顯示3-MA可能藉由對PI3K的抑制作用而抑制Akt，並經PI3K/Akt影響到TLR4的訊息傳遞。由於GSK3b是Akt下游的一個重要受質，過去也發現該蛋白分子可正調控NF-kB活性及NF-kB所轉錄之細胞激素的生成，因此我們也進一步探討3-MA對於GSK3b的調控作用。實驗結果發現3-MA促進NF-kB活化、iNOS與IL-1b的表現，及抑制IFNb的表現與STAT1活化等作用皆會受GSK3b抑制劑(SB216763與SB415286)所回復。然而，3-MA抑制IL-10和IL-6的表現作用卻不受GSK3b抑制劑所影響。最後，我們進一步證實了3-MA在我們的系統裡扮演著誘發細胞自噬的角色。3-MA會增加LC3-I轉換成LC3-II，並且促進autolysosome的生成。此外，以siRNA干擾Atg5表現後發現3-MA原先促進NF-kB活化的作用會消失。總結地說，利用3-MA當作研究工具，我們發現Akt和GSK3b會共同參與調控發炎反應和細胞自噬的生成。

關鍵字

脂多醣；巨噬細胞；發炎；細胞自噬

並列摘要

3-Methyladenine (3-MA) is one of the most well known pharmacological inhibitors of autophagy commonly applied in autophagy researches today. However, rather than inhibiting class III PI3K that is thought to result in autophagy suppression, 3-MA has also been demonstrated to interfere with class I PI3K and consequently augment autophagy flux. To date, not only many have queried about the pharmacological specificity of this chemical compound, controversies have also arisen over the virtual action mechanisms of 3-MA in the recent years. There is an intimate interplay between autophagy and inflammation, and 3-MA may exert various off-target effects that are closely associated with inflammation beyond autophagy issue. In this study, we aim to get a thorough understanding on the action mechanisms of 3-MA in toll-like receptor 4 (TLR4)-mediated inflammatory responses, and moreover, to decipher the action of 3-MA in modulation of autophagy. In this respect, signaling pathways downstream of PI3K/Akt/GSK3b were investigated in 3-MA-treated murine RAW264.7 macrophages upon LPS stimulation. We found that 3-MA could enhance various LPS-induced inflammatory responses including NF-kB activation, iNOS, COX-2 and pro-inflammatory cytokines IL-1b and IL-12 production. In contrast, 3-MA suppressed LPS-induced production of IFNb, IL-10 and IL-6. Studies on the upstream TLR4 signaling revealed that 3-MA can positively regulate TAK1, p38, JNK, p65, but negatively regulate TBK1 and IRF3. Akt, one of the major downstream signaling molecules of class I PI3K that is in close relationship with TLR4, has been demonstrated to play a dual role differentially to regulate MyD88- and TRIF-dependent signaling pathways. Our results showed that 3-MA can inhibit Akt as a result of class I PI3K interference, and implicated the contribution of PI3K/Akt in 3-MA-regulated upstream signaling of TLR4. As GSK3b is an important Akt substrate that can also regulate NF-kB activation as well as cytokine production, we further explored its involvement in the actions of 3-MA. We found that 3-MA-induced NF-kB activation, iNOS and IL-1b expression as well as inhibition of IFNb expression and STAT1 activation upon LPS stimulation can be reversed by the GSK3b inhibitors (SB216763 and SB415286). However, 3-MA-induced inhibition of IL-10 and IL-6 production failed to be affected by SB216763. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in our system, as LC3-I conversion to LC3-II and appearance of autolysosome were both induced. Moreover, increasing NF-kB activity by 3-MA was no longer observed in LPS-stimulated macrophages with siAtg5 treatment. This study using 3-MA as a tool shed new insight into molecular events that Akt and GSK3b work cooperatively in orchestrating inflammatory responses and autophagy formation.

並列關鍵字

LPS ； macrophage ； inflammation ； autophagy

參考文獻

Akira, S. (2006). TLR signaling. Curr Top Microbiol Immunol 311, 1-16.

Akira, S., and Takeda, K. (2004). Toll-like receptor signalling. Nat Rev Immunol 4, 499-511.

Aksoy, E., Vanden Berghe, W., Detienne, S., Amraoui, Z., Fitzgerald, K.A., Haegeman, G., Goldman, M., and Willems, F. (2005). Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4. Eur J Immunol 35, 2200-2209.

Androulidaki, A., Iliopoulos, D., Arranz, A., Doxaki, C., Schworer, S., Zacharioudaki, V., Margioris, A.N., Tsichlis, P.N., and Tsatsanis, C. (2009). The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs. Immunity 31, 220-231.

Anjum, R., and Blenis, J. (2008). The RSK family of kinases: emerging roles in cellular signalling. Nat Rev Mol Cell Biol 9, 747-758.

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3-Methyladenine調控脂多醣在巨噬細胞活化之發炎反應的機制探討

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