- 學位論文
可溶性腺苷酸環化酶(soluble adenylyl cyclase, sAC)在調控mTORC1訊息傳遞途徑的角色探討
The role of soluble adenylyl cyclase (sAC) in regulating mTORC1 signaling pathway
摘要
在進行增生的細胞中，麩醯胺酸依賴的回補作用(Glutamine-dependent anaplerosis)對於維持提供細胞生長所需巨分子生合成前驅物的cataplerosis活性扮演一個中心的功能角色。然而，此麩醯胺酸依賴的回補作用在調控主要細胞生長調節途徑mTOR signaling pathway以及對於細胞命運(細胞複製或細胞老化)的抉擇上的影響，還尚未清楚。另外，越來越多的證據顯示，粒線體代謝途徑的狀態影響細胞生長調節者mTORC1的活性。近來，可溶性腺苷酸環化酶(soluble adenylyl cyclase)被認為是一個粒線體TCA cycle的感知者，可以感知粒線體TCA cycle的狀態，且可進而調節粒線體ATP合成的效率。在本研究中，我們利用amino-oxyacetate (AOA)，一個轉胺酶抑制物，可以抑制麩醯胺酸依賴的回補作用。在人類纖維母細胞(WI38 cells)，AOA處理造成mTORC1/2活性平衡改變(mTORC1失活、mTORC2活化)，並且在時間數據上的比較分析上，此活性的起始變化並非導因於細胞內的ATP耗盡或是ADP:ATP比值上升。另外，AOA誘導的mTORC2活化，並非透過S6K1對於Rictor的Thr1135磷酸化負回饋機制控制；但可能與Rictor的蛋白質總量上升相關，但仍需進一步分析。另外，雖然粒線體reactive oxygen species (ROS)在失能的粒線體誘發細胞老化或凋零的機制上，扮演著角色。但在AOA處理下的狀態下，人類的纖維母細胞(WI38 cells)並未觀察到明顯的細胞內ROS量的改變；並且，AOA直接與ROS scavenger N-乙醯半胱氨酸(N-acetyl-cysteine)共同處理，無法抑制AOA誘發的細胞老化或是細胞複製。顯示AOA抑制麩醯胺酸依賴的回補作用所誘發的細胞老化反應不是經由ROS的訊號媒介產生。AOA誘發的細胞老化反應具有兩個階段，前期可逆細胞週期停滯階段，此期間只要恢復提供麩醯胺酸依賴的回補作用，細胞會重新進入細胞複製週期；後期則為不可逆細胞老化週期終止階段，只要通過某一個轉折期，即便在恢復提供麩醯胺酸依賴的回補作用，細胞也不會恢復細胞複製。時間上的分析顯示，p16INK4A可能在不可逆期扮演著重要的角色。 此外，為了探討可溶性腺苷酸環化酶是否參與在麩醯胺酸依賴的回補作用媒介的mTORC1活性中，我們利用可溶性腺苷酸環化酶(soluble adenylyl cyclase)的選擇性專一性抑制物KH7來抑制可溶性腺苷酸環化酶的活性。在人類纖維母細胞(WI38 cells)或人骨肉瘤細胞(U2OS cells)，處理KH7同樣會造成mTORC1/2活性平衡改變(mTORC1失活、mTORC2活化)，並且進一步造成ATP下降，粒線體群體模式改變及崩解以及細胞生長受到抑制。透過starvation-refeeding的處理方式，KH7在時間上對於麩醯胺酸依賴的回補作用媒介的mTORC1活性，有不同效應: 短期KH7處理(15分鐘)會增強麩醯胺酸依賴的回補作用媒介的mTORC1活性；然而長期KH7處理(120分鐘)反而會抑制麩醯胺酸依賴的回補作用媒介的mTORC1活性。另外，碳酸酐酶抑制劑(acetazolamide)只會造成增強麩醯胺酸依賴的回補作用媒介的mTORC1活性。顯示，可溶性腺苷酸環化酶影響mTORC1活性可能透過不同機制。 本研究顯示，麩醯胺酸依賴的回補作用的狀態除了對於可進行複製細胞的細胞命運(cell fate)扮演角色之外，更提供一個可能的熱點使得mTORC1可以更精確的衡量感知細胞即時的氨基酸可得性(amino acid availability)狀態。而在其中，可溶性腺苷酸環化酶(soluble adenylyl cyclase)可能扮演著重要的角色，媒介粒線體代謝活性與mTOR signaling。
並列摘要
In proliferating cells, glutamine-dependent anaplerosis plays a central role to support cataplerosis-mediated efflux of precursors derived from the TCA cycle intermediates for biomass accumulation, yet its role on central cell growth mTOR signaling pathway and on cell fate determination remain poorly understood. In the present study, we found that sustained blockade of glutamine-dependent anaplerosis with amino-oxyacetate (AOA), a transaminase inhibitor which targets glutaminolytic pathways, leading to mTORC1 inactivation and mTORC2 activation is not due to the ATP depletion, no significant effects on the intracellular ATP contents were observed for as long as 2 days of AOA treatment, whereas the mTORC1 activity was rapidly reduced in WI38 cells within 12 hours of the treatment. Moreover, AOA-induced mTORC2 was not mediated by blocking S6K1 activity toward to the inhibitory phosphorylation of Rictor at Thr1135. In contrast, we observed the increased rictor protein level upon AOA treatment, but its correlation with mTORC2 requires further investigation. Although production of ROS by the mitochondria is known to be a critical senescence-inducing factor, our results suggest that AOA did not increase the intracellular ROS levels and co-treatment with the ROS scavenger N-acetyl-cysteine (NAC) failed to block the AOA-induced growth inhibition and cellular senescence. Moreover, we observed that AOA-induced senescence program is a two stages senescence response: a transient reversible cell cycle arrest before about 3 days of AOA treatment, and subsequent an irreversible senescent growth arrest after 4 days of treatment, similar to replicative senescence. On the other hand, recent studies implicate that soluble adenylyl cyclase (sAC), a special member of adenylyl cyclase family that generates classic secondary messenger cAMP, serves as a sensor of the TCA cycle modulating the efficiency of ATP synthesis, implicating its correlation with glutamine-dependent anaplerosis and mTOR pathways. We observed that inhibition of sAC with KH7 led to mTORC1 inactivation, mTORC2 activation, ATP depletion, collapse of mitochondrial network, and proliferation inhibition in U2OS cells, implicating the essential role of sAC activity for mitochondrial physiology as well as cell growth. Of note, we found that short-term KH7 treatment enhanced glutamine-dependent anaplerosis driven mTORC1 activation, while long-term KH7 treatment still significantly inhibited mTORC1 activity even in the presence of glutamine and leucine.