探討Fas Ligand經由非依賴型凋亡蛋白酵素所誘發之細胞死亡以及發炎反應的訊息傳遞路徑

Abstract

Fas是死亡受體的原型，當它活化的時候可以直接活化凋亡蛋白酵素，最終導致各種細胞凋亡的產生。Fas所傳達的細胞凋亡分子機轉已經被研究得非常透徹；但是，除了引發細胞凋亡之外，最近的研究顯示：在細胞凋亡的路徑被抑制的時候，Fas也可以引起一些非細胞凋亡的訊息傳遞路徑。目前，Fas所傳導的這種替代性訊息傳遞路徑的分子機轉仍然不甚清楚。在本研究當中，我們利用小鼠胚胎纖維母細胞來探討Fas所傳達的凋亡蛋白酵素非依賴型細胞死亡的訊息傳遞路徑，以及這種傳遞路徑和發炎反應之間的相關性。我們發現，在小鼠胚胎纖維母細胞中，Fas ligand所引起的細胞死亡會因為前處理廣效型凋亡蛋白酵素抑制劑z-VAD-FMK後而更為增強。我們的結果顯示RIP和TRAF2在這種凋亡蛋白酵素非依賴型細胞死亡中扮演了很重要的角色。這種凋亡蛋白酵素非依賴型的細胞死亡也和細胞內活性氧化物（ROS）的累積相關，而活性氧化物的清除劑butylated hydroxyanisol (BHA)則可以預防這種的細胞死亡。此外，我們還觀察到延遲且持續的JNK和NF-kB的活化，及粒腺體膜電位損壞，這些現象也都可以利用前處理BHA而消失。我們也發現在前處理z-VAD-FMK後， FasL除了會引起凋亡蛋白酵素非依賴型的細胞死亡，同時也可以引起強烈的發炎反應，包括造成COX-2以及IL-8表現量增加，而這個發炎反應是和持續的JNK以及NF-kB的活化有關。總結來說，我們闡述Fas ligand在小鼠胚胎纖維母細胞所誘發的非依賴凋亡蛋白酵素的訊息傳遞路徑，會導致細胞的死亡和發炎反應的發生。

Fas is the prototype of the death receptors which can directly activate caspases and eventually lead to apoptosis in various cell types. The molecular mechanism of Fas-mediated apoptosis has been well investigated. However, besides apoptosis, recent studies showed that Fas can induce some non-apoptotic signaling pathways under conditions where apoptosis pathways have been inhibited. Nowadays, the molecular mechanism of these alternative signaling pathways mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-mediated caspase-independent cell death and the coupling of this alternative cell death to inflammatory responses in mouse embryonic fibroblasts (MEFs). We found that in MEFs, FasL induced cell death was sensitized under pre-treatment of pan-caspase inhibitor z-VAD-FMK. Our results revealed that RIP and TRAF2 may play important roles in FasL-induced caspase-independent cell death. This death is also associated with intracellular reactive oxygen species (ROS) accumulation, as the ROS scavenger butylated hydroxyanisol (BHA) can prevent the cell death. Furthermore, delayed and sustained JNK, NF-kB activation, and mitochondrial membrane potential breakdown were observed. These phenomena can be eliminated by pre-treatment of BHA. We also found that in company with caspase-independent cell death, Fas ligand can also induce strong inflammatory responses including COX-2 and IL-8 gene up-regulation. This inflammatory response is associated with the delayed and sustained JNK and NF-kB activation. Taken together, we demonstrate caspase-independent signaling pathway of Fas in MEF, and show its activation lead to cell death and inflammatory responses.