RNA腺苷脱氨酸ADAR2在胰岛β-细胞中功能与调控的机制研究
文献类型：学位论文
| 作者 | 杨柳 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-05 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 刘勇 |
| 关键词 | RNA编辑 可调节性细胞分泌 葡萄糖刺激的胰岛素分泌 JNK |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 哺乳动物中介导的A至I RNA编辑的RNA腺苷脱氨酶主要由ADAR1和ADAR2两个家族成员组成，它们具有编辑下游底物神经递质受体和离子通道并改变其活性的功能，从而在中枢神经系统中发挥着极其重要的作用, 但是到目前为止，对于ADAR2在外周组织的其他生理学功能仍知之甚少。我们前期的实验表明，ADAR2所介导的RNA编辑在胰岛β细胞中受到营养能量代谢的调控，为了探寻ADAR2在胰岛β细胞和其他专职分泌细胞中的作用，我们利用RNA干扰技术特异性地降低 ADAR2的表达，发现ADAR2的表达降低后可显著性地抑制INS-1β细胞系和体外分离的小鼠胰岛中葡萄糖刺激的胰岛素分泌。此外，为了研究ADAR2对于细胞分泌的影响是否仅限于胰岛β细胞，我们检测了在神经嗜铬细胞瘤PC12细胞中ADAR2对于KCl诱导的内源嗜铬粒B蛋白的分泌和外源转入的人生长激素的分泌的影响。结果表明，ADAR2在Neuron-like 细胞可调节性分泌中同样起到了重要的作用。值得注意的是，回复性表达具有编辑活性的ADAR2可以修正ADAR2的降低对于可调节性细胞分泌削弱的影响，而回复性表达不具有编辑活性的ADAR2则不能呈现此效应。此外，ADAR2表达量的减少伴随着的葡萄糖刺激的膜融合的减少和停靠在细胞膜边缘分泌囊泡数目的降低；而与之相关联的是囊泡分泌相关的重要分子，如Munc18-1和Synaptotagmin-7的蛋白表达均出现一定程度的下降。综上所述，我们揭示了迄今从未报道过的腺苷脱氨酶ADAR2在可调节性细胞分泌中的重要调控作用，并且暗示着RNA编辑作为一种全新的机制调控细胞分泌的关键环节。 目前，关于ADAR2基因表达调控的研究鲜有报道。我们前期的工作初步证明了葡萄糖可以刺激胰岛β细胞中ADAR2的表达，但是其具体的调控机制尚不明了。因此，我们用浓度逐渐升高的葡萄糖刺激INS-1β细胞，结果显示，ADAR2的蛋白表达和JNK的磷酸化水平的上升均呈现浓度依赖性。进而，我们用JNK的抑制剂抑制JNK分子的活性和腺病毒干扰系统特异的降低JNK1的表达，发现这两种干预方式均会极大程度上削弱葡萄糖刺激的ADAR2的表达上升。另一方面，为了研究JNK对于ADAR2的调控是普遍存在于不同细胞类型还是特异的存在于胰岛β细胞，我们检测了在小鼠胚胎纤维原野生型细胞、JNK1表达缺失型细胞和JNK2表达缺失型细胞中内源ADAR2的表达。 结果表明在JNK1表达缺失型细胞中几乎检测不到ADAR2的mRNA和蛋白的表达，而JNK2表达缺失型细胞则未出现此类现象。最后，我们分析了在JNK1和JNK2全身敲除小鼠的不同组织，如胰岛、肝脏和下丘脑中的ADAR2的表达变化，结果显示，ADAR2的mRNA表达仅在JNK1，而不是JNK2，敲除小鼠的胰岛中特异性降低。综上所述，JNK1是胰岛β细胞中能量代谢情况下调控ADAR2表达的关键分子，而且我们的研究结果预示着ADAR2所介导的RNA编辑和JNK1所介导的细胞压力应答可能存在着功能上的重要关联。 |
| 索取号 | D2010-051 |
| 英文摘要 | Mammalian RNA editing catalyzed by adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2, plays pivotal roles in the brain through functional modifications of neurotransmitter receptors and ion channels. We have previously demonstrated that RNA editing by ADAR2 is metabolically regulated in pancreatic β-cells. To investigate the cellular functions of ADAR2 in β-cells and other professional secretory cells, we studied the effects of ADAR2 knockdown on regulated exocytosis. We found that selective knockdown of ADAR2 expression not only markedly impaired glucose-stimulated insulin secretion in the rat insulinoma INS-1 cells and isolated primary pancreatic islets, but also significantly diminished the secretion of exogenous human growth hormone or endogenous chromogranin B protein upon KCl stimulation in the rat adrenal pheochromocytoma PC12 cells. Importantly, restored overexpression of the catalytically active but not editing-deficient mutant ADAR2 was able to correct the impairment in stimulated secretion from ADAR2 knockdown cells. Moreover, suppression of ADAR2 expression significantly attenuated Ca2+-evoked membrane capacitance increases and appreciably reduced the number of membrane-docked insulin granules in INS-1 cells. Interestingly, the secretory defects caused by ADAR2 deficiency were coupled to decreased expression of Munc18-1 and synaptotagmin-7, two key molecules in the regulation of vesicle exocytosis. Thus, these findings reveal an important aspect of ADAR2 actions in regulated exocytosis, implicating RNA editing as a novel mechanism in the control of cellular secretory machinery. While our previous studies showed that ADAR2 in pancreatic islets and β-cells was regulated by nutritional and energy status, the molecular mechanisms by which ADAR2 expression was induced remains largely unclear. To investigate further the key players that are involved in glucose stimulation of ADAR2 expression, we found that in parallel with dose-dependent stimulation by glucose of ADAR2 expression,phosphorylation of the 46-and 54-kDa isoforms of c-Jun NH2-terminal kinase (JNK) was concurrently induced in insulin-secreting INS-1 cells. Supporting the idea that JNK contributes to the regulation of ADAR2 expression and ADAR2-mediated RNA editing, inhibition of JNK with an inhibitor or knockdown with shRNA of the expression of JNK1 prominently suppressed glucose-augmented ADAR2 expression as well as its editing activity in INS-1 cells. Furthermore, neither the mRNA abundance nor the protein level of ADAR2 was detectable in JNK1-deficient MEF cells as compared to that of WT or JNK2-deficient MEF cells, indicating the specific requirement of JNK1 for ADAR2 transcription. Consistently, we observed that the mRNA expression of ADAR2 was selectively reduced in the islets of JNK1 null mice in comparison with that of WT or JNK2 null mice. Taken together, these data demonstrate that in pancreatic islets and β-cells JNK1 serves as a crucial mediator in response to nutritional stimulation in the regulation of ADAR2 expression. This also suggests that ADAR2-mediated RNA editing may be functionally connected to JNK1-dependent cellular stress pathways. |
| 语种 | 中文 |
| 公开日期 | 2015-12-24 |
| 源URL | [http://202.127.25.144/handle/331004/315]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_糖脂代谢与调控研究组 |
| 推荐引用方式 GB/T 7714 | 杨柳. RNA腺苷脱氨酸ADAR2在胰岛β-细胞中功能与调控的机制研究[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2010. |
入库方式： OAI收割
来源：上海营养与健康研究所
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