【作者】 杨俊；

【导师】 刘继红； 李本义；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 糖原合酶激酶3β(Glycogen synthase kinase 3beta, GSK-3β)是一种遗传保守的丝氨酸／苏氨酸蛋白激酶,在细胞周期代谢、基因转录、细胞凋亡、细胞运动、肿瘤发生等过程中发挥重要调控作用。研究表明,GSK-3β在众多的细胞生命活动中均处于一定程度的持续激活状态,尤其在细胞饥饿条件下,其活性显著增强,表明GSK-3p对于维持饥饿状态的细胞存活具有重要意义,但目前对于GSK-3β调控细胞存活／死亡的机制仍不清楚。本研究旨在通过抑制处于不同生长环境的前列腺癌细胞的GSK-3β活性,观察其对细胞存活／死亡的影响,鉴别并探讨GSK-3β对细胞自噬、凋亡及坏死等不同死亡模式的调控作用,初步阐明饥饿条件下,GSK-3β调控细胞存活／死亡的分子途径。结果表明,在正常培养条件下(10%FBS),通过药物或靶向siRNA抑制GSK-3β活性后,前列腺癌细胞并未发生明显的死亡反应；然而在血清饥饿条件下,抑制GSK3β却导致明显的细胞坏死反应,而且进一步研究发现,该坏死过程中伴随着细胞自噬反应的显著增强。由于细胞饥饿状态本身可诱导一定程度的自噬反应,为探讨细胞自噬在GSK-3β失活诱导的细胞坏死过程中的作用,进一步的实验观察了饥饿条件下,细胞自噬与GSK-3β活性均被抑制后的细胞反应,实验结果显示,细胞自噬被抑制时,GSK-3β失活诱导的细胞死亡却并未消失,而是由坏死反应转化为凋亡反应。为阐明上述GSK-3β活性抑制导致的不同死亡反应的分子机制,本研究对不同细胞死亡通路(自噬,坏死,凋亡)的关键调控分子的蛋白表达变化进行了探讨,结果发现,饥饿状态下抑制前列腺癌细胞GSK-3β活性,可时间／剂量依赖性地上调Bif-1蛋白的表达,促进Bif-1与beclin-1及VPS34形成自噬复合体,增强细胞自噬反应；但在正常培养条件下,Bif-1蛋白表达水平却无明显变化；当同时抑制自噬反应及GSK-3β活性时,凋亡调控因子Bax蛋白的活化结构Bax(6A7)明显增强。然而当抑制Bif-1基因表达后,不同条件下抑制GSK-3β活性导致的细胞自噬、坏死或凋亡等反应均消失,相应条件下,Bax活化明显降低。在进一步的核酸水平研究中发现,抑制GSK-3β活性可明显增强Bif-1基因启动子活性,导致Bif-1mRNA水平上调。综上研究结果表明,GSK-3β通过Bif-1调控前列腺癌细胞的死亡通路。在饥饿状态下,抑制GSK-3β活性可上调Bif-1蛋白表达,诱导细胞发生广泛的自噬反应,最终导致细胞坏死发生,当上述过程中细胞自噬被抑制时,Bif-1转而激活Bax,导致细胞凋亡发生。更多还原

【Abstract】 Glycogen synthase kinase 3β(GSK-3β) belongs to a family of conserved serine/threonine kinases present in all eukaryotic groups. It was shown to function in a wide range of biological processes, including cell cycle progression, gene transcription, apoptosis, cellular metabolism, cell movement and tumorigenesis. Emerging evidence revealed that GSK-3βis constantly active in cells and its activity increases significantly after serum deprivation, indicating that GSK-3βmay play a major role in cell death/survival under serum starvation. In the present study, we attempted to understand how GSK-3βcontrols cell death/survival after serum depletion. Under full culture conditions(10% FBS), GSK-3βinhibition with chemical inhibitors or siRNA failed to induce cell death in human prostate cancer cells. By contrast, under conditions of serum starvation, a profound necrostic cell death was observed as evidenced by cellular morphologic features and biochemical markers. Further analysis revealed that GSK-3β-inhibition-induced cell death was parallel with an extensive autophagic response. Interestingly, blocking the autophagic response did not block GSK-3β-inhibition-induced cell death but switched it from necrosis to apoptotic cell death, which was associated with a Bax conformational change. Most importantly, knocking down bif-1 expression abolished GSK-3β-inhibition-induced autophagic response and cell death, as well as lysosomal membrane permeabilization, indicating that bif-1 is requires for the GSK-3β-inhibition-induced cytotoxic effect. Finally, GSK-3βsuppression increased bif-1 promoter activity, and subsequently up-regulated bif-1 expression, enhanced bif-1 interaction with Beclin-1/vps34 complex that is a key component in the autophagic process. Take together, our data suggest that GSK-3βcontrols cell death/survival by modulating of bif-1 dependent autophagic reponse and bif-1 dependent bax conformational change.更多还原