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論文名稱(外文):Role of histone deacetylase 6 on the activation of calcium store sensor STIM1
指導教授(外文):Meng-Ru Shen
外文關鍵詞:Ca2+SOCESTIM1HDAC6microtubulecervical cancer
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細胞內鈣離子的恆定對於調節細胞之生理功能例如細胞增生、遷移及基因表現扮演重要的角色。鈣池調控鈣離子流入(SOCE),是在非興奮性細胞中造成鈣離子進入細胞並穩定細胞內鈣離子訊號的機制,基質交互分子(STIM1)是位於內質網上的鈣離子感測蛋白,當內質網中的鈣離子濃度下降時,STIM1會被活化並聚集,並且和鈣池調控鈣離子通道蛋白Orai1產生交互作用,進而啟動SOCE。先前研究指出,微管在SOCE活化過程中及調控STIM1在細胞內分佈位置扮演重要的角色。組蛋白去乙醯酶6 (HDAC6)可將微管去乙醯化,並改變微管之穩定性,且已知內質網的型態是由微管間交互作用來維持。我的研究目標在於探討HDAC6在STIM1活化過程中扮演的角色。我利用了藥理和基因方法來研究HDAC6對於STIM1在細胞中的移動、STIM1與微管尖端蛋白EB1的交互作用以及對於SOCE的影響。由免疫螢光染色的結果顯示,HDACs抑制劑並不會影響Thapsgargin (TG)引起的STIM1活化與聚集。在活化STIM1之後,我分析STIM1在細胞內的分佈及微管乙醯化程度,發現只有抑制HDAC家族中的HDAC6可影響STIM1由內質網移動到細胞膜邊的過程。另外,由單細胞鈣離子濃度分析的結果也顯示HDAC6抑制劑可阻斷SOCE的活化,並與劑量呈現正相關的趨勢。重要的是,給予正常的子宮頸上皮細胞tubastatin-A並不會增加微管的乙醯化,也不影響其SOCE的活化。綜合以上實驗結果,證明HDAC6在子宮頸癌細胞的STIM1活化過程中及鈣離子的流入扮演重要的角色,並可能提供予未來臨床應用。
Intracellular calcium (Ca2+) homeostasis is important for many cellular functions such as proliferation, migration and gene expression. Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway and plays an important role in generation of the sustained Ca2+ signals in non-excitable cells. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor which is essential for SOCE. Upon Ca2+ depletion from ER stores, STIM1 aggregates into multiple puncta and translocates to the plasma membrane, where STIM1 interacts with Orai1 to mediate Ca2+ entry. Previous studies showed that microtubules play a facilitative role in SOCE activation by optimizing the localization of STIM1. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase that can deacetylate microtubules and can alter the stability of microtubules. It is well established that the morphology of ER is maintained through a tight interaction with microtubules. My study aims to investigate the role of microtubule-associated deacetylase HDAC6 on the activation of ER Ca2+ store sensor STIM1. I used a combination of pharmacological and genetic approaches to investigate the roles of HDAC6 on the membrane trafficking of STIM1, the interaction of STIM1 with microtubule-binding protein EB1 and SOCE activation. Results from immunofluorescent images showed that all HDAC inhibitors did not interfere with the formation of STIM1 multiple puncta induced by thapsigargin. I analyzed the distribution of STIM1 and acetylated-α-tubulin after STIM1 activation. Only inhibition of HDAC6 can interfere STIM1 trafficking from ER to plasma membrane. Consistently, the results of single cell intracellular Ca2+ measurement showed that HDAC6 inhibitors blocked the SOCE activity in a dose-dependent manner. In contrast, the treatment of tubastatin-A did not affect the acetylation of -tubulin and the activity of SOCE in the normal cervical cells. Taken these results together, I suggest that HDAC6 plays an important role in STIM1 trafficking and further affects Ca2+ influx in cervical cancer cells. This study makes HDAC6 a potential therapeutic target in cervical cancer.
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