mTORCs 參與 aminooxyacetate 抑制粒線體之 malate-aspartate shuttle 所引發的早發性細胞老化

Abstract

細胞老化是一種細胞週期的現象，細胞老化的生物意義可能是做為個體老化的細胞基礎以及抑癌作用。鑑於粒線體在個體老化上扮演的重要角色，例如，自由基傷害累積理論等，而且粒線體所調節的營養以及代謝反應對於細胞生長週期也有決定性的影響，因此我們參考胰臟β細胞的粒線體對細胞營養狀態的感知而刺激胰島素分泌的系統，試驗 AOA 抑制 malate-aspartate shuttle 後，對細胞生長週期的反應。本實驗室過去發現AOA會誘發正常人類胚胎肺臟纖維母細胞株 WI38 細胞老化，而且 AOA 對細胞造成的老化效應可以被 5 mM αKG (α-ketoglutarate) 所阻斷，本論文進一步發現 0.3 mM 的非必需氨基酸（NEAA）亦可阻斷AOA的細胞老化效應，而且AOA所誘發的老化細胞呈現縮狹的非典型形態。我們指出此種類型的早發性細胞老化和粒線體有所相關，但與 H2O2 產生無關。AOA 作用會使得mTORC1活性消失，mTORC2的活性增加，而此種效應在 αKG 的添加後回復正常；但是 NEAA 的添加卻使得 mTORC1 的活性輕微的上升以及 mTORC2 的活性微幅的降低，且細胞的形態沒有回復正常。另外也發現到在AOA處理下，細胞內的 mTOR 會在細胞質內相對累積。同樣的，mTORC1 活性的消失也反應在受到 mTOR 所正向調控 HIF-1α 轉譯方面。除了 HIF-1α 轉譯受到 mTORC1 的正向調節外，其 HIF-1α 的堆積也受到 hydroxylase 對其做的 hydroxylation 之影響。然而，此 hydroxylase 屬於需要 αKG 做為輔受質的 dioxygenase，而且從果蠅的研究中發現，此 hydroxylase 會促進細胞生長週期的運轉。因為 αKG 的添加對AOA效應之阻斷，我們推測 AOA 對細胞老化的誘發來自於粒線體內 αKG 的不足，進而抑制細胞內此 hydroxylase 原本具有促進細胞生長的角色，最後導致細胞邁向老化階段。本研究透過在 AOA 所誘發老化以及可被 αKG 或 NEAA 添加後而阻斷其老化之系統中所得的結果顯示，粒線體可能透過調節 mTORCs 活性影響細胞生長週期，並且可能成為誘發細胞老化的根源。在個體的老化的粒線體理論，提供了有別於自由基傷害累積理論的新觀點。

It is known that mitochondria play an important role in organism aging, such as free radical damage theory. Mitochondria also play a critical role in integrating nutrient information and metabolic activity during cell growth. In order to study the mitochondrial role in cell senescence, we refer to mitochondria as a nutrient sensor in pancreatic β cells, and have established a cell senescence system induced by aminooxyacetate (AOA) inhibition of mitochondrial malate-aspartate shuttle in normal human embryonic lung fibroblasts WI38, and we showed that α-ketoglutarate (αKG) blocked AOA-induced cell senescence. In this study, we further demonstrate that non-essential amino acid (NEAA) also blocked the AOA-induced cell senescence, but the cells still displayed an atypical (non-spreading) senescent morphology. This premature senescence is related to mitochondrial function, and independent of peroxide production. Interestingly, we observed that AOA treatment resulted in a dramatic reduction of mTORC1 activity, and the simultaneous appearance of mTORC2 hyperactivity. αKG blocked the AOA-induced imbalance of mTORC1 and mTORC2 activity. Although similar effect as αKG blockade, NEAA effect on mTORC1 and mTORC2 was relatively weaker. We also observed mTOR predominantly localized in the cytoplasm under AOA treatment. Decreasing αKG could lead to inactivation of the αKG-dependent dioxygenases, including PHD (prolyl-4-hydroxylase-domain protein). PHD regulates the stabilization of HIF-1αand promotes cell growth in the fly. Because αKG blocks AOA effect, we speculated that AOA-induced cell senescence was resulted from mitochondrial αKG insufficiency that subsequently inhibited the positive role of the hydroxylase in cell growth cycle, and eventually led to cell senescence. Taken together, our results demonstrate αKG and NEAA blocked AOA-induced senescence and relative activity shift between mTORC1 and mTORC2 in normal human embryonic fibroblasts. This suggests that mitochondria could control cell growth cycle via regulation of mTORCs activity, and this could be the key that trigger cell senescence. Furthermore, our studies provide a normal viewpoint regarding mitochondrial role in organism aging besides the theory of accumulation of free radical-induced damage.