Regulation of cellular growth in adverse environmental conditions

不利环境条件下细胞生长的调节

• 批准号: RGPIN-2016-04752
• 负责人: Gupta, Madhulika
• 金额: $ 2.26万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=652705
• 关键词: Regulation; cellular; growth; adverse; environmental

OBJECTIVE: The Long Term Goal of my NSERC-funded research program is to gain a basic understanding of the underlying mechanisms that regulate cellular proliferation in adverse environments. The overall objectives of this research are to determine the O2 sensing mechanism/s controlled by hypoxia and how they affect IGF-dependent cell proliferation.****BACKGROUND: The mechanistic target of rapamycin (mTOR) integrates signals from O2 to positively regulate cell growth. Hypoxia decreases cell proliferation by repressing mTOR signaling. Insulin-like growth factor I (IGF-I) is a peptide that promotes cell proliferation. A link between hypoxia and IGF-I signaling exists, but a link of IGF-I signaling with mTOR is unclear. Hypoxia induces IGF binding protein-1 (IGFBP-1), a key negative regulator of IGF-I action. Phosphorylation converts IGFBP-1 into a bio-inhibitor of IGF-I action. The mechanisms that regulate IGFBP-1 phosphorylation in hypoxia and inhibit IGF action are largely uncharacterized.****PROGRESS AND JUSTIFICATION: Findings from my NSERC-funded research have put-forth a novel concept that hypoxia triggers site-specific IGFBP-1 hyperphosphorylation to inhibit cell proliferation. We have shown that mTOR is an important regulator of IGF-I action. My 2014 NSREC-funded data suggest that hypoxia induced IGFBP-1 phosphorylation may be linked with mTOR. I hypothesize that hypoxia inhibits mTOR, which activates protein kinase CK2 for hypoxia-induced IGFBP-1 phosphorylation and inhibition of IGF-I-action. I will use HepG2 cells and techniques e.g., RNAi/immunoblotting/MRM-MS and functional proteomics to test my hypothesis in 3 Aims.*******Aim1. Determine that mTORC1 is required in regulating hypoxia-induced IGFBP-1 phosphorylation. We will identify upstream activators of mTORC1 signaling using by siRNA silencing to show the requirement of mTORC1 inhibition in IGFBP-1 phosphorylation and reduction of cell growth via IGF-I action.*******Aim 2. Determine the role of CK2 in IGFBP-1 phosphorylation. We will use multiple strategies such as co-immunoprecipitation, phospho-site mapping and a CK2 inhibitor resistant mutant to determine that IGFBP-1 is bona fide CK2 substrate.*******Aim 3. Determine that CK2 is regulated by mTORC1 in hypoxia. We will silence CK2 with/without mTORC1/C2 silencing in normoxia or hypoxia to determine whether mTOR regulates CK2 expression/activity and IGF-I bioactivity in hypoxia.*******SIGNIFICANCE: This research will provide fundamental knowledge of the interactive cellular mechanisms involving mTOR and CK2 that control IGF action in hypoxia, critical for developmental, physiological, and aberrant processes.*******HQP: Trainees in my laboratory will receive a strong background in the fundamentals of protein biochemistry, functional-proteomics, and molecular-cell biology to achieve recognition/scholarships and a career in academia/industry/governmental laboratories.***

目的：我的NSERC资助的研究计划的长期目标是对在不利环境中调节细胞增殖的潜在机制有一个基本的了解。本研究的总体目标是确定缺氧控制的O2传感机制/S及其如何影响依赖胰岛素样生长因子的细胞增殖。*背景：雷帕霉素的机制靶点整合来自O2的信号，正向调节细胞生长。低氧通过抑制mTOR信号通路抑制细胞增殖。胰岛素样生长因子I(IGF-I)是一种促进细胞增殖的多肽。低氧与IGF-I信号之间存在联系，但IGF-I信号与mTOR之间的联系尚不清楚。低氧诱导胰岛素样生长因子结合蛋白-1(IGFBP-1)，它是IGF-I作用的关键负性调节因子。磷酸化将IGFBP-1转化为IGF-I作用的生物抑制物。在低氧中调节IGFBP-1磷酸化和抑制IGF作用的机制在很大程度上尚不清楚。*进展和理由：我的NSERC资助的研究发现提出了一个新的概念，即低氧触发位点特异性的IGFBP-1过度磷酸化以抑制细胞增殖。我们已经证明mTOR是IGF-I作用的重要调节因子。我2014年NSREC资助的数据表明，低氧诱导的IGFBP-1磷酸化可能与mTOR有关。我假设低氧抑制mTOR，mTOR激活蛋白激酶CK2，使低氧诱导的IGFBP-1磷酸化，并抑制IGF-I-作用。我将使用HepG2细胞和技术，例如RNAi/免疫印迹/MRM-MS和功能蛋白质组学来验证我的假设在三个目标。确定mTORC1在调节缺氧诱导的IGFBP-1磷酸化过程中是必需的。我们将通过siRNA沉默来确定mTORC1信号的上游激活物，以表明抑制mTORC1在IGFBP-1磷酸化中的必要性，并通过IGF-I作用抑制细胞生长。目的2.确定CK2在IGFBP-1磷酸化中的作用。我们将使用免疫共沉淀、磷酸化位点定位和抗CK2抑制剂突变体等多种策略来确定IGFBP-1是真正的CK2底物。目的3.确定CK2在缺氧时受mTORC1调控。我们将在常氧或低氧条件下沉默CK2，以确定mTOR是否调节CK2的表达/活性和IGF-I的生物活性。*意义：这项研究将提供涉及mTOR和CK2的相互作用的细胞机制的基础知识，这些机制控制着IGF在低氧中的作用，对发育、生理和异常过程至关重要。*HQP：我实验室的学员将获得蛋白质生物化学、功能蛋白质组学和分子细胞生物学的坚实背景，以获得认可/奖学金，并在学术界/工业/政府实验室获得职业生涯。**