Cracking the signaling code: how ERK activity dynamics coordinate gene expression and differentiation in keratinocytes

破解信号密码：ERK 活性动态如何协调角质形成细胞中的基因表达和分化

• 批准号: 9759329
• 负责人: Siddhartha Jena
• 金额: $ 4.5万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9759329
• 关键词: Behavior; Biochemical; Biological Markers; Calcium; Cell Culture Techniques; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cell division; Cells; Code; Complex; Cues; Cyclin D1; Data; Development; Dissection; ERG gene; Early Gene Transcriptions; Engineering; Epithelial; Event; Extracellular Signal Regulated Kinases; Foundations; Frequencies; Gene Expression; Gene Expression Regulation; Gene Family; Genes; Genetic Transcription; Goals; Growth Factor; Hour; Image; Immediate-Early Genes; Light; Link; MAP Kinase Signaling Pathways; Maintenance; Maps; Measures; Monitor; Mus; Nature; Organ; Output; Pathway interactions; Patients; Physiologic pulse; Physiological; Proliferating; Publishing; Regenerative Medicine; Regulation; Regulator Genes; Reporter; Reporter Genes; Research Personnel; Role; Serum; Signal Pathway; Signal Transduction; Skin; Skin graft; Starvation; Stem cells; Synthetic Genes; System; Testing; Tissue Engineering; Transcription Factor AP-1; Undifferentiated; Work; cell behavior; cell type; cellular imaging; experimental study; feeding; filaggrin; insight; involucrin; keratinocyte; keratinocyte differentiation; live cell imaging; loricrin; optogenetics; programs; promoter; response; three dimensional cell culture; tissue-level behavior; unpublished works

Project Summary: The development and maintenance of epithelial organs on a cellular level is reliant upon concerted networks of biochemical signaling pathways, which transmit environmental cues such as growth factor and calcium concentrations to the level of gene expression in the cell. One example is basal keratinocytes, which have important proliferative and differentiation roles in the body. The precise regulation of these behaviors establishes the spatial axis of differentiated cells that makes up the skin. Both proliferative and differentiation cell behaviors have been linked to Ras/ERK MAP kinase pathway signaling; however, the precise mechanisms through which the same pathway regulates both responses remain unclear. Moreover, with an increasing body of work suggesting that epidermal progenitor cells may someday be used to culture patient-specific skin grafts in a dish, an understanding of single-cell signaling dynamics becomes increasingly important in dissecting the emergent tissue level behavior. Our lab has introduced live reporters of ERK activity into primary mouse keratinocytes and found that ERK demonstrates rich, complex endogenous dynamics. The objective of this proposal is to understand if and how Ras/ERK pathway dynamics in keratinocytes are read out at the level of cellular behavior, resulting in either proliferation or differentiation. I will use a combination of live cell reporter imaging, single-cell imaging of transcriptional dynamics, and engineering of synthetic reporter genes to dissect how ERK activity dynamics feed into the ERK-downstream gene expression program. To control physiologically relevant ERK behavior, I will use a combination of organotypic cell culture and optogenetic inputs. These approaches will be used in concert to probe input-output functions of ERK dynamics to gene expression, to test the hypothesis that different dynamic ERK behaviors are read out in different gene expression programs. A successful completion of the proposed work will constitute the first detailed dissection of ERK dynamics in a primary cell context as well as mapping of these dynamics to outputs in the form of physiologically relevant gene expression and cell responses. A comprehensive understanding of live signaling dynamics in differentiating, proliferating skin will likely have a broad impact on tissue engineering, regenerative medicine, and a deeper fundamental understanding of epithelial organs.

项目概要： 上皮器官在细胞水平上的发育和维持依赖于协调一致的网络 生化信号通路，传递生长因子和钙等环境信号 浓度与细胞内基因表达水平有关。一个例子是基底角质形成细胞，它具有 在体内具有重要的增殖和分化作用。对这些行为的精准监管 建立构成皮肤的分化细胞的空间轴。增殖和分化 细胞行为与 Ras/ERK MAP 激酶通路信号传导有关；然而，精确的机制 同一途径调节这两种反应的机制仍不清楚。而且随着身材的不断增加 大量工作表明表皮祖细胞有一天可能用于培养患者特异性皮肤移植物 在培养皿中，对单细胞信号传导动力学的理解在剖析单细胞信号传导过程中变得越来越重要。 紧急组织水平行为。我们的实验室已将 ERK 活动的实时报告引入原代小鼠中 角质形成细胞，发现 ERK 表现出丰富、复杂的内源动力学。此举的目的 提议是了解角质形成细胞中的 Ras/ERK 通路动态是否以及如何在 细胞行为，导致增殖或分化。我将使用活细胞报告器的组合 成像、转录动力学的单细胞成像以及合成报告基因的工程分析 ERK 活性动态如何进入 ERK 下游基因表达程序。控制 生理相关的 ERK 行为，我将结合使用器官型细胞培养和光遗传学 输入。这些方法将共同用于探测 ERK 动态对基因的输入输出功能 表达，以检验不同基因中读出不同动态 ERK 行为的假设 表达程序。成功完成拟议工作将构成第一次详细剖析 原代细胞环境中 ERK 动态的分析，以及将这些动态映射到以下形式的输出： 生理相关的基因表达和细胞反应。对实时信号的全面了解 分化、增殖皮肤的动态可能会对组织工程、再生产生广泛影响 医学，并对上皮器官有更深入的基础了解。