Repurposing classical death pathways for signaling roles in lens differentiation

重新利用经典死亡途径在晶状体分化中发挥信号作用

• 批准号: 9187024
• 负责人: Marc Kantorow
• 金额: $ 54.21万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187024
• 关键词: Area; Autophagocytosis; Autophagosome; Binding; Biogenesis; Biological Assay; CASP3 gene; Cell Cycle; Cell Death; Cell Differentiation process; Cell Maturation; Cells; Cessation of life; Chick Embryo; Chromatin; Cleaved cell; Complex; Crystallins; Cytoplasm; DNA; DNA Binding; DNA strand break; Data; Degenerative Disorder; Development; Developmental Process; Disease; Down-Regulation; E-Cadherin; Eating; Elements; Embryo; Ensure; Epithelial Cells; Event; Excision; Exhibits; FYCO1 gene; Gamma-H2AX; Gene Deletion; Gene Expression; Human; Image; Impairment; Laboratories; Lens Fiber; Lens development; Light; Logic; Lysosomes; MAPK8 gene; Mediating; Methods; Microtubules; Mitochondria; Monitor; Mutation; Natural regeneration; Nucleosomes; Organ; Organ Culture Techniques; Organelles; Outcome Study; Pathway interactions; Pattern; Phosphorylation; Play; Positioning Attribute; Process; Proteins; Regulation; Regulator Genes; Regulatory Pathway; Repression; Retina; Role; Science; Signal Pathway; Signal Transduction; Stress; Techniques; Testing; Tissues; Transport Vesicles; Undifferentiated; Vesicle Transport Pathway; Vision; Work; alpha-Crystallins; caspase-activated deoxyribonuclease; chromatin remodeling; congenital cataract; cytochrome c; fiber cell; genetic regulatory protein; inhibitor/antagonist; innovation; lens; lens transparency; novel; prevent; programs; public health relevance; response; spatiotemporal; success; transcriptome; transcriptome sequencing

 DESCRIPTION (provided by applicant): This application will establish new pathways required for initiation of lens cell differentiation and will reveal a long-sought after mechanism for lens organelle elimination upon lens cell maturation to achieve lens transparency. We have recently discovered that low-level caspase-3 activation is required for initiation of lens cell differentiaton and that a new macroautophagy pathway initiated by JNK down-regulation directs organelle elimination during lens cell maturation. AIM 1 will establish a novel function for α-crystallin in regulating mitochondrial cytochrome c release to ensure low-level caspase-3 activation required for lens cell differentiation. AIM 2 will establish how low-level caspase-3 activity transmits its differentiation initiation signal through activation of caspase 3-dependent DNAase (CAD) and Mst1 (and its downstream effector γH2AX) to direct chromatin remodeling events that we propose are required for lens cell differentiation. AIM 3 will functionally define the macroautophagy proteins and pathways that direct the temporal and spatial removal of organelles during lens cell maturation. The logic, feasibility, and potential success of these AIMs is supported by our strong preliminary data demonstrating that: α-crystallin translocates to the mitochondria where it complexes with cytochrome c to modulate its release and likely ensure low-level caspase-3 activation required for initiation of lens cell differentiation; CAD is activatd and H2AX phosphorylated and bound to DNA in a caspase-3-dependent manner during initiation of lens cell differentiation likely to initiate DNA strand breaks and γH2AX DNA binding leading to nucleosome positioning changes required for initiation of lens differentiation- specific gene expression; and key macroautophagy regulatory proteins exhibit expression patterns consistent with their functioning in the JNK-regulated MTORC1 pathway that leads to removal of lens cell organelles upon lens cell maturation. The AIMs proposed are significant because their testing will establish new mechanisms crucial to regulation of the decision of lens cells to begin their differentiation program, as well as developmental processes critical for lens transparency. The work will be applicable towards understanding the differentiation, development and disease states of other tissues since the regulatory molecules examined are common to many other tissues. The proposed AIMs are conceptually innovative since they establish new roles and requirements for classic cell death regulators in normal signaling roles in the cell that regulate differentiation and development. The AIMs are technically innovative since they employ sophisticated lens organ culture methods, gene expression and deletion strategies, signaling assays and chromatin monitoring techniques developed and established in our laboratories.

 描述（由申请人提供）：本申请将建立启动透镜细胞分化所需的新途径，并将揭示长期寻求的在透镜细胞成熟后消除透镜细胞器以实现透镜透明度的机制。我们最近发现，低水平的caspase-3激活是启动透镜细胞分化所必需的，并且JNK下调启动的新的宏自噬途径指导了透镜细胞成熟期间细胞器的消除。AIM 1将为α-晶体蛋白在细胞内的功能研究提供新的思路。 调节线粒体细胞色素C释放以确保透镜细胞分化所需的低水平半胱天冬酶-3活化。AIM 2将确定低水平的caspase-3活性如何通过激活caspase-3依赖性DNA酶（CAD）和Mst 1（及其下游效应物γ H2 AX）来传递其分化起始信号，以指导我们提出的透镜细胞分化所需的染色质重塑事件。AIM 3将在功能上定义宏观自噬蛋白和途径，指导透镜细胞成熟期间细胞器的时间和空间去除。这些目标的逻辑、可行性和潜在成功 我们强有力的初步数据支持：α-晶状体蛋白易位至线粒体，在线粒体中与细胞色素c复合以调节其释放，并可能确保启动透镜细胞分化所需的低水平caspase-3活化; CAD被激活，H2 AX被磷酸化并在半胱天冬酶-3-磷酸化酶中与DNA结合。在透镜细胞分化启动过程中可能启动DNA链断裂和γ H2 AX DNA结合的依赖性方式 导致启动透镜特异性分化所需的核小体定位变化 基因表达;并且关键的大自噬调节蛋白表现出与它们在JNK调节的MTORC 1途径中的功能一致的表达模式，所述MTORC 1途径导致在透镜细胞成熟时除去透镜细胞器。所提出的AIM是重要的，因为它们的测试将建立对调节透镜细胞开始其分化程序的决定至关重要的新机制，以及对透镜透明度至关重要的发育过程。这项工作将适用于了解其他组织的分化，发育和疾病状态，因为检查的调节分子对许多其他组织是共同的。所提出的AIM在概念上是创新的，因为它们为经典细胞死亡调节剂在调节分化和发育的细胞中的正常信号传导作用建立了新的作用和要求。AIM在技术上具有创新性，因为它们采用了我们实验室开发和建立的复杂的透镜器官培养方法、基因表达和缺失策略、信号分析和染色质监测技术。