Role of the Snail1-Twist-p21 axis on cell cycle arrest and renal fibrosis development

Snail1-Twist-p21 轴在细胞周期停滞和肾纤维化发展中的作用

基本信息

批准号：
10062964
负责人：
Jianhua Xing
金额：
$ 34.2万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-10 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062964
关键词：
Acute Renal Failure with Renal Papillary Necrosis Address Adopted Affect Apoptosis Area Biology Cell Cycle Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Line Cells Cellular biology Chronic Kidney Failure Clustered Regularly Interspaced Short Palindromic Repeats Color Cost of Illness Coupled Coupling Data Data Analyses Development Dialysis procedure Dimensions Drug Targeting Effectiveness Elements End stage renal failure Epithelial Cells Evaluation Event Extracellular Matrix Fibrosis Flow Cytometry Fluorescence Foundations Future G2/M Arrest Genes Germ Cells Goals Image Image Analysis Individual Kidney Kidney Transplantation Label M cell Maps Mathematics Measurable Measurement Medicare Methods Modeling Monitor Normal tissue morphology Pathologic Patients Periodicity Phenotype Play Population Positioning Attribute Probability Process Property Proteins Research Research Design Role Seminal Series Structure System Systems Biology Testing Time Tubular formation Up-Regulation Validation automated image analysis base cellular imaging deep learning deep learning algorithm effective therapy epithelial to mesenchymal transition established cell line experimental study global health human old age (65+)imaging study kidney epithelial cell kidney fibrosis live cell imaging loss of function mathematical analysis mathematical model network models new therapeutic target novel programs quantitative imaging renal epithelium success therapy design therapy development tool transcription factor treatment strategy

项目摘要

Chronic kidney diseases (CKD) such as kidney fibrosis are global health challenges. In the US alone CKD cost Medicare an estimated 50 billion dollars for patients with CKD age 65 and older in 2013. Recent studies revealed that after AKI renal epithelial cells undergo a partial epithelial-to-mesenchymal transition (pEMT) and G2/M cell cycle arrest through a Snail1-Twist1-p21 axis; these cells secrete profibrotic factors and contribute to fibrosis progression. Therefore these three factors become promising potential drug targets for treating fibrosis, but further development requires addressing several outstanding open questions. The temporal sequence and causal relation between pEMT and cell cycle arrest is controversial, and the respective roles of Snail1, Twist1, and p21 on regulating pEMT and cell cycle arrest is unclear. Addressing these questions requires quantitative systems biology approaches beyond cell biology methods traditionally used in the field. In recent years my lab has made progression on deep learning based image automated analysis for live cell images, CRISPR-based gene editing, and mathematical modeling and other quantitative biology tools. These technological developments position us to tackle the above-mentioned challenging questions related to kidney fibrosis. Based on existing studies and our preliminary results, we hypothesize that there is a temporal order of the three factors, with p21 initializing G2/M arrest, which is reinforced by subsequent upregulation of Snail1; Snail1 also activates, and Twist1 further maintains the pEMT program; due to their temporally varying roles, effectiveness of targeting these factors depends on the timing of treatment. We will test the hypothesis with quantitative imaging studies using established cell lines and primary renal epithelial cells and mathematical analysis of competing models. In Aim 1, we will perform multi-color flow cytometry studies and time-lapse imaging studies on progression of cell cycle, EMT, and other cell fates of cells under stimulation. The two types of studies will provide complementary information on whether pEMT and cell cycle are tightly coupled, and will map out the temporal sequence of events of various cell fate change as well as correlation to expression levels of the three factors. In Aim 2, we will monitor the temporal profiles of these factors through fluorescence protein tagging in single cells, and use the data to evaluate an ensemble of models to identify one or a set of minimal network regulating EMT and G2/M arrest. We will then further examine the roles of individual factors through model analysis and a series of inhibition experiments. Success of the proposed research will provide mechanistic understanding of the regulatory network of cell cycle arrest and EMT in renal epithelial cells. The proposed research is our starting point for an emerging field of quantitative systems biology on kidney fibrosis. We expect that introducing quantitative approaches will greatly accelerate future development of treatment strategies on the increasing global health challenge imposed by progression of fibrosis, which currently lacks effective treatment.

肾纤维化等慢性肾脏疾病（CKD）是全球健康挑战。仅在美国 CKD 成本 2013 年，估计为 65 岁及以上 CKD 患者提供 500 亿美元的医疗保险。最近的研究揭示 AKI 后肾上皮细胞经历部分上皮间质转化 (pEMT) G2/M 细胞周期通过 Snail1-Twist1-p21 轴停滞；这些细胞分泌促纤维化因子并有助于纤维化进展。因此，这三个因素成为治疗纤维化有希望的潜在药物靶点，但进一步的发展需要解决几个悬而未决的问题。时间顺序和 pEMT 与细胞周期停滞之间的因果关系存在争议，Snail1、Twist1、 p21 和 p21 对 pEMT 和细胞周期停滞的调节作用尚不清楚。解决这些问题需要定量系统生物学方法超越了该领域传统使用的细胞生物学方法。近年来我的实验室在基于深度学习的活细胞图像自动分析、基于 CRISPR 的图像自动分析方面取得了进展基因编辑、数学建模和其他定量生物学工具。这些技术进展使我们能够解决上述与肾纤维化相关的挑战性问题。根据现有的研究和我们的初步结果，我们假设存在一个时间顺序三个因素，p21 启动 G2/M 期停滞，随后 Snail1 的上调增强了这一作用；蜗牛1 也激活，Twist1进一步维持pEMT程序；由于他们的角色随时间变化，针对这些因素的有效性取决于治疗的时机。我们将检验假设使用已建立的细胞系和原代肾上皮细胞进行定量成像研究和数学竞争模型分析。在目标 1 中，我们将进行多色流式细胞术研究和延时对刺激下细胞周期、EMT 和其他细胞命运进展的成像研究。两种类型多项研究将提供关于 pEMT 和细胞周期是否紧密耦合的补充信息，并将绘制各种细胞命运变化事件的时间顺序以及与表达水平的相关性三个因素中。在目标 2 中，我们将通过荧光蛋白监测这些因素的时间分布在单个细胞中进行标记，并使用数据来评估模型集合，以识别一个或一组最小的调节 EMT 和 G2/M 逮捕的网络。然后，我们将通过以下方式进一步研究各个因素的作用：模型分析和一系列抑制实验。该研究的成功将为细胞调控网络提供机制上的理解肾上皮细胞周期停滞和 EMT。拟议的研究是我们新兴领域的起点肾纤维化的定量系统生物学。我们预计引入定量方法将大大加快针对日益严峻的全球健康挑战的未来治疗策略的制定纤维化进展造成的，目前缺乏有效的治疗方法。