The Akt Pathway in Regulation of H. pylori-induced TRAIL-mediated Apoptosis in Human Gastric Epithelial Cells

胃幽門螺旋桿菌 (Helicobacter pylori, HP) 主要分布於人類的腸胃道中，是目前最常見的人類病原菌之一，根據世界衛生組織的調查，現今大約有百分之五十的全球人口受其感染，研究也指出感染胃幽門螺旋桿菌和慢性胃炎、胃潰瘍以及胃癌均有十分密切的關聯性。根據前人的研究發現，胃部上皮細胞的大量死亡是感染胃幽門螺旋桿菌的重要指標，另外T淋巴球會在感染時浸潤至發炎區域，並且表現可以誘導細胞凋亡的TRAIL蛋白質。在我們實驗室發表的研究論文顯示，胃幽門螺旋桿菌能夠藉由負調控short form FLICE-inhibitory protein (FLIPS)蛋白質的表現量，進而增強形成TRAIL死亡誘導訊息複合物 (Death-Inducing Signaling Complex, DISC) 導致caspase-8的活化，來影響人類胃部上皮細胞對於TRAIL的敏感性。除此之外，實驗室的初步實驗也指出感染胃幽門螺旋桿菌會降低胃上皮細胞內Akt的磷酸化，且和FLIPS表現量具有一致性。但是，Akt相關的訊息傳遞路徑究竟是如何調節胃幽門螺旋桿菌所造成的TRAIL敏感性，目前還不清楚。 在此論文中，我們探討了Akt訊息傳遞路徑在胃幽門螺旋桿菌誘發TRAIL媒介的胃上皮細胞凋亡之角色，首先，在實驗結果中顯示，Akt的磷酸化以及FLIPS表現量會受到和胃幽門螺旋桿菌的共培養而下降，同時此現象有時間依賴性。另外，不管是使用siRNA抑制Akt的表現量，或用負調控Akt磷酸化的上游分子PI3K的化學抑制物，亦或是利用質體轉染過量表現Akt分子於胃上皮細胞株中，都能夠觀察到FLIPS表現量和Akt的磷酸化具有正向相關性。此外，過度表現Akt於細胞中還能有效抑制胃幽門螺旋桿菌誘發TRAIL媒介的胃上皮細胞凋亡。 根據曾經發表過的期刊論文，Akt-USP8-AIP4-FLIPS訊息傳遞路徑參與在調控FLIPS降解，進而影響TRAIL誘導的細胞凋亡，而我們的研究也發現，這條路徑也參與在幽門螺旋桿菌誘發TRAIL媒介的胃上皮細胞凋亡中，當Akt的磷酸化下降，USP8作為一個去泛素化酵素會增進AIP4的活性，而AIP4則是泛素化酵素會使FLIPS的泛素化增加，而使其降解，導致細胞凋亡的增加。 除了研究細胞端的訊息分子，延續實驗室之前的初步結果，我們發現到CagE對於胃幽門螺旋桿菌誘發TRAIL媒介的胃上皮細胞凋亡相當重要，而非毒素CagA，當使用了剔除CagE的突變菌株，以上所發現的訊息傳遞路徑菌不會造成改變。 綜上所論，這些結果有助於加深對胃幽門螺旋桿菌造成細胞凋亡之致病機制的認識，同時提供作為臨床治療的可能方向。Helicobacter pylori (H. pylori) is one of the most common human pathogens which can be found in the upper gastrointestinal tract. There are about 50% of global population infected, and H. pylori infection is associated with chronic gastritis, peptic ulcer and even gastric cancer. It has been reported that the apoptosis of gastric epithelial cells infected by H. pylori is increased and contributes to the pathogenesis of gastric diseases. Previous studies have shown that, during H. pylori infection, gastric-infiltrating T cells are increased and express TRAIL which is capable of inducing apoptosis. Our lab has reported that human gastric epithelial cells sensitized to H. pylori are conferred susceptibility to TRAIL-mediated apoptosis by enhanced assembly of the TRAIL death-inducing signaling complex (DISC) and caspase-8 activation through down-regulation of the short form of cellular FLICE-inhibitory protein (FLIPS). In the preliminary data, we have found that H. pylori infection suppressed Akt phosphorylation correlated to decreased FLIPS expression. However, the role of Akt pathway in regulating H. pylori-induced TRAIL-mediated apoptosis remains unclear. In this thesis, we investigated how Akt pathway in gastric epithelial cells is affected by H. pylori followed the altered sensitivity to TRAIL. First, the results indicated that the phosphorylation of Akt and expression of FLIPS are reduced in a time-dependent manner in the presence of H. pylori. We also demonstrated that the activation of Akt is positively correlated to the expression level of FLIPS in AGS cell line through either Akt knockdown by siRNA, Akt inactivation by PI3K inhibitor or Akt overexpression by transfection. Furthermore, H. pylori-induced TRAIL-mediated apoptosis was inhibited by Akt overexpression. Following the proposed mechanism involved in FLIPS degradation through Akt-USP8-AIP4-FLIPS pathway in TRAIL-induced apoptosis, our data revealed that, during H. pylori infection, phosphor-Akt downregulation increased ubiquitin-specific protease 8 (USP8) expression, which decreased the ubiquitination of the FLIPS E3 ubiquitin ligase, atrophin interacting protein 4 (AIP4). More active AIP4 caused the enhanced ubiquitination and degradation of FLIPS and apoptosis induction. In addition, we have examined different upstream molecules of Akt and found that PTEN phosphorylation decreased after H. pylori infection. Moreover, from the aspect of pathogen, we have found that one of the proteins of cag pathogenicity island, CagE, but not CagA, played an important role in altering host cells sensitivity to TRAIL in the preliminary data. In consistent with this finding, the expression of FLIPS and USP8 and the activation of Akt cannot be altered by △CagE or △CagPAI mutant infection. Taken together, this study gives us an insight into the underlying mechanism of H. pylori-induced TRAIL-mediated apoptosis and provides the possible candidate molecules for the therapeutic target in H. pylori-associated gastric disease