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: 9054227
• 负责人: Marc Kantorow
• 金额: $ 55.42万
• 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/9054227
• 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; Gene Deletion; Gene Expression; Human; Image; 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; Staging; Stress; Techniques; Testing; Tissues; 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(申请人提供)：这项申请将建立启动晶状体细胞分化所需的新途径，并将揭示一种长期追求的机制，即晶状体细胞成熟后消除晶状体细胞器，以实现晶状体透明。我们最近发现，低水平的caspase-3激活是启动晶状体细胞分化所必需的，并且JNK下调启动的一条新的宏观自噬途径指导晶状体细胞成熟过程中细胞器的消除。目标1将为α建立一个新的功能-晶状体蛋白 调节线粒体细胞色素c的释放，以确保晶状体细胞分化所需的低水平caspase-3的激活。目的研究低水平的caspase-3活性如何通过激活caspase-3依赖的DNA酶(CAD)和Mst1(及其下游效应因子γH_2AX)来传递其分化起始信号，从而指导我们提出的晶状体细胞分化所需的染色质重塑事件。目标3将从功能上定义大自噬蛋白和途径，指导晶状体细胞成熟过程中细胞器的时空移除。这些目标的逻辑、可行性和潜在的成功 我们的初步数据表明：α-晶体蛋白被转移到线粒体，在那里它与细胞色素c形成络合物以调节其释放，并可能确保启动晶状体细胞分化所需的低水平的caspase-3的激活；在晶状体细胞分化的启动过程中，cad被激活，h2ax被磷酸化，并以caspase-3依赖的方式与dna结合，可能启动dna链断裂和γh2ax dna结合。 导致启动晶状体特异性分化所需的核小体位置改变 基因表达；关键的宏自噬调节蛋白表现出与它们在JNK调节的mTORC1途径中的功能一致的表达模式，该途径导致晶状体细胞成熟时细胞器的去除。提出的目标意义重大，因为他们的测试将建立新的机制，对调控晶状体细胞开始分化计划的决定至关重要，以及对晶状体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，因为所研究的调节分子在许多其他组织中是共同的。提出的目标在概念上是创新的，因为它们为经典的细胞死亡调节因子在调节细胞分化和发育的正常信号作用中建立了新的角色和要求。这些目标具有技术创新性，因为它们使用了我们实验室开发和建立的复杂的晶状体器官培养方法、基因表达和缺失策略、信号分析和染色质监测技术。