SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 10000726
• 负责人: ALAN R KIMMEL
• 金额: $ 138.3万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10000726
• 关键词: Ablation; Adipocytes; Adipose tissue; Affect; Agonist; Anabolism; Bacteria; Binding Proteins; Biochemical; Biogenesis; Biological Assay; Biological Models; Biology; Cardiac; Cardiac Myocytes; Cardiac Output; Cardiovascular Diseases; Cell Adhesion; Cell Communication; Cell Cycle; Cell Density; Cell Fate Control; Cells; Characteristics; Chemotactic Factors; Chemotaxis; Chronic; Communication; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Defense Mechanisms; Development; Dictyostelium; Dictyostelium discoideum; Discrimination; Disease; Embryonic Development; Essential Genes; Eukaryota; Event; Excision; Exhibits; Family; Fatty Acids; Fatty Liver; Food; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genome; Glucose; Gram-Negative Bacteria; Growth; Growth and Development function; Hamartoma; Heart; Human; Hydrolysis; Hypertriglyceridemia; Immunologics; Individual; Inflammation; Inflammatory; Insulin Resistance; Knockout Mice; Life Cycle Stages; Ligands; Lipids; Lipolysis; Membrane; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Modeling; Molecular Genetics; Movement; Mus; Muscle Fibers; Mutation Analysis; Myocardial; Myocardial Infarction; Myocardial dysfunction; Natural Immunity; Neoplasm Metastasis; Nutrient; Nutrient Depletion; Organelles; Organism; Pathologic Processes; Pathway interactions; Pattern; Peroxisome Proliferator-Activated Receptors; Phagocytes; Phagocytosis; Phase; Phospholipids; Phosphotransferases; Physiological Processes; Play; Population; Positioning Attribute; Process; Program Development; Protein Biosynthesis; Proteins; Receptor Activation; Receptor Signaling; Regulation; Reporting; Ribosomes; Risk Factors; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sirolimus; Source; Starvation; Stimulus; Stress; Stroke Volume; Surface; System; Techniques; Time; Triglycerides; Up-Regulation; Variant; Wild Type Mouse; Withdrawal; Wound Healing; amplification detection; cell growth; cell growth regulation; cell motility; density; energy balance; fMet-Leu-Phe receptor; fatty acid oxidation; fibroblast growth factor 21; heart function; human disease; intercellular communication; interest; lipid metabolism; mitochondrial membrane; molecular modeling; non-oncogenic; novel; overexpression; oxidation; pathogen; perilipin; perilipin A; prevent; programs; protective factors; rapid growth; response; sensor; steroid hormone; trafficking; tumorigenesis; uncoupling protein 1; uptake

Kinases mTORC1 and AMPK act as energy sensors, controlling nutrient responses and cellular growth. Changes in nutrient levels affect diverse transcriptional networks, making it challenging to identify downstream paths that regulate cellular growth or a switch to development via nutrient variation. The life cycle of Dictyostelium presents an excellent model to study the mTORC1 signaling function for growth and development. Dictyostelium grow as single cells in nutrient-rich media, but, upon nutrient withdrawal, growth ceases and cells enter a program for multi-cell development. While nearly half the genome shows gene expression changes upon nutrient removal, we hypothesized that not all of these genes are required for the switch to program development. Through manipulation of mTORC1 activity alone, without nutrient removal, we focused on a core network of genes that are required for switching between growth and development for regulation of cell fate decisions. To identify developmentally essential genes, we sought ways to promote development in the absence of nutrient loss. We first examined the activities of mTORC1 and AMPK in Dictyostelium during phases of rapid growth and starvation-induced development and showed they exhibited reciprocal patterns of regulation under various conditions. Using these as initial readouts, we identified rich media conditions that promoted rapid cell growth but, upon mTORC1 inactivation by rapamycin, led to a growth/development switch. Examination of gene expression during cell fate switching showed that changes in expression of most starvation-regulated genes were not required for developmental induction. Approximately 1000 genes which become downregulated upon rapamycin treatment comprise a cellular growth network involving ribosome biogenesis, protein synthesis, and cell cycle processes. Conversely, the upregulation of 500 genes by rapamycin treatment defines essential signaling pathways for developmental induction, and 135 of their protein products intersect through the well-defined cAMP/PKA network. Many of the rapamycin-induced genes we found are currently unclassified, and mutation analyses of 5 such genes suggest a novel gene class essential for developmental regulation. We show that manipulating activities of mTORC1/AMPK in the absence of nutrient withdrawal is sufficient for a growth-to-developmental fate switch in Dictyostelium, providing a means to identify transcriptional networks and signaling pathways essential for early development. Initial immunological defense mechanisms to pathogen invasion rely on innate pathways of chemotaxis and phagocytosis, original to ancient phagocytes. Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Although chemotaxis has been well-studied in mammalian and model systems using purified chemoattractants in defined conditions, directed movement toward live bacteria has been more difficult to assess. Dictyostelium discoideum is a professional phagocyte that chemotaxes toward bacteria during growth-phase in a process to locate nutrient sources. Using Dictyostelium as a model, we have developed a system that is able to quantify chemotaxis to very high sensitivity. Here, Dictyostelium can detect various chemoattractants at concentrations <1 nM. Given this exceedingly sensitive signal response, Dictyostelium will migrate directionally toward live gram positive and gram negative bacteria, in a highly quantifiable manner, and dependent upon bacterially-secreted chemoattractants. Additionally, we have developed a real-time, quantitative assay for phagocytosis of live gram positive and gram negative bacteria. To extend the analyses of endocytic functions, we further modified the system to quantify cellular uptake via macropinocytosis of smaller (<100 kDa) molecules. These various approaches provide novel means to dissect potential for identification of novel chemoattractants and mechanistic factors that are essential for chemotaxis, phagocytosis, and/or macropinocytosis and for more detailed understanding in host-pathogen interactive defenses. Cell-cell interactions and response are enhanced by increased cell density. We were interested to identify novel secreted proteins that accumulate in parallel to the collective local cell population and that can direct developmental decisions. We chose Dictyostelium, which grow as individual cells in rich nutrient sources, but initiate a multi-cell developmental program as nutrients become depleted. Cell density sufficiency is critical to multi-cell formation in Dictyostelium, and we hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at specific cell-density thresholds. We have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development, to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression become centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell autonomous functions for regulation of cellular adhesion and growth. Myocardial dysfunction is commonly associated with accumulation of cardiac lipid droplets (LDs). Perilipin 2 (Plin2) is a LD protein that is involved in LD formation, stability and trafficking events within the cell. Even though Plin2 is highly expressed in the heart, little is known about its role in myocardial lipid storage. A recent report shows that cardiac overexpression of Plin2 result in massive myocardial steatosis suggesting that Plin2 stabilizes LDs. In this study, we hypothesized that deficiency in Plin2 would result in reduced myocardial lipid storage. In contrast to our hypothesis, we found increased accumulation of triglycerides in hearts, and specifically in cardiomyocytes, from Plin2-/- mice. Although Plin2-/- mice had markedly enhanced lipid levels in the heart, they had normal heart function under baseline conditions and under mild stress. However, after an induced myocardial infarction, stroke volume and cardiac output were reduced in Plin2-/- mice compared with Plin2+/+ mice. We further demonstrated that the increased triglyceride accumulation in Plin2-deficient hearts was caused by altered lipophagy. Together, our data show that Plin2 is important for proper hydrolysis of LDs. Beige adipocytes can dissipate energy as heat. Elaborate communication between metabolism and gene expression is important in the regulation of beige adipocytes. Although lipid droplet (LD) binding proteins play important roles in adipose tissue biology, it remains unknown whether perilipin 3 (Plin3) is involved in the regulation of beige adipocyte formation and thermogenic activities. In this study, we demonstrate that Plin3 ablation stimulates beige adipocytes and thermogenic gene expression in inguinal white adipose tissue (iWAT). Compared with wild-type mice, Plin3 knockout mice were cold tolerant and displayed enhanced basal and stimulated lipolysis in iWAT, inducing peroxisome proliferator-activated receptor (PPAR) activation. In adipocytes, Plin3 deficiency promoted PPAR target gene and uncoupling protein 1 expression and multilocular LD formation upon cold stimulus. Moreover, fibroblast growth factor 21 expression and secretion were upregulated, which was attributable to activated PPAR in Plin3-deficient adipocytes. These data suggest that Plin3 acts as an intrinsic protective factor preventing futile beige adipocyte formation by limiting lipid metabolism and thermogenic gene expression.

激酶 mTORC1 和 AMPK 充当能量传感器，控制营养反应和细胞生长。营养水平的变化会影响不同的转录网络，因此很难确定调节细胞生长或通过营养变化转向发育的下游路径。盘基网柄菌的生命周期为研究生长和发育的 mTORC1 信号传导功能提供了一个极好的模型。盘基网柄菌在营养丰富的培养基中以单细胞形式生长，但是，在营养撤除后，生长停止并且细胞进入多细胞发育程序。虽然近一半的基因组显示营养物去除后基因表达发生变化，但我们假设并非所有这些基因都是转向程序开发所必需的。通过单独操纵 mTORC1 活性而不去除营养，我们专注于在生长和发育之间切换以调节细胞命运决定所需的核心基因网络。为了确定发育必需基因，我们寻找在不损失营养的情况下促进发育的方法。我们首先检查了盘基网柄菌在快速生长和饥饿诱导发育阶段中 mTORC1 和 AMPK 的活性，并表明它们在各种条件下表现出相互的调节模式。使用这些作为初始读数，我们确定了丰富的培养基条件可以促进细胞快速生长，但在雷帕霉素使 mTORC1 失活后，会导致生长/发育转换。对细胞命运转换过程中基因表达的检查表明，大多数饥饿调节基因的表达变化并不是发育诱导所必需的。大约 1000 个在雷帕霉素处理后下调的基因组成了涉及核糖体生物合成、蛋白质合成和细胞周期过程的细胞生长网络。相反，雷帕霉素治疗对 500 个基因的上调定义了发育诱导的重要信号通路，其中 135 个蛋白质产物通过明确的 cAMP/PKA 网络相交。我们发现的许多雷帕霉素诱导基因目前尚未分类，对 5 个此类基因的突变分析表明，存在一种对于发育调控至关重要的新基因类别。我们发现，在没有营养撤退的情况下，操纵 mTORC1/AMPK 的活性足以实现盘基网柄菌从生长到发育的命运转换，从而提供了一种识别早期发育所必需的转录网络和信号通路的方法。 针对病原体入侵的初始免疫防御机制依赖于源自古代吞噬细胞的先天趋化和吞噬作用途径。趋化性和细胞迁移在胚胎发生、炎症和伤口愈合等正常生理过程以及慢性炎症性疾病和癌症转移等病理过程中发挥着关键作用。尽管在特定条件下使用纯化的化学引诱剂在哺乳动物和模型系统中对趋化性进行了充分研究，但对活细菌的定向运动更难以评估。盘基网柄菌是一种专业的吞噬细胞，在生长阶段对细菌进行趋化，以定位营养源。使用盘基网柄菌作为模型，我们开发了一种能够以非常高的灵敏度量化趋化性的系统。在这里，盘基网柄菌可以检测浓度 <1 nM 的各种化学引诱剂。鉴于这种极其敏感的信号响应，盘基网柄菌将以高度可量化的方式定向向活革兰氏阳性和革兰氏阴性细菌迁移，并依赖于细菌分泌的化学引诱剂。此外，我们还开发了一种实时定量测定活革兰氏阳性和革兰氏阴性细菌的吞噬作用。为了扩展内吞功能的分析，我们进一步修改了系统，通过较小（<100 kDa）分子的巨胞饮作用来量化细胞摄取。这些不同的方法提供了新的方法来剖析鉴定对趋化性、吞噬作用和/或巨胞饮作用至关重要的新型化学引诱剂和机制因素的潜力，并为更详细地了解宿主-病原体相互作用防御提供了新的方法。 细胞密度的增加增强了细胞间的相互作用和反应。我们有兴趣识别新的分泌蛋白，这些蛋白与集体局部细胞群并行积累，并且可以指导发育决策。我们选择了盘基网柄菌，它在丰富的营养源中作为单个细胞生长，但当营养耗尽时启动多细胞发育程序。细胞密度充足对于盘基网柄菌的多细胞形成至关重要，我们假设新型分泌蛋白可能作为密度传感因子，在特定的细胞密度阈值下促进多细胞发育命运决定。我们纯化了一种新型分泌蛋白 DPF，它充当发育的密度感应因子，并定义盘基网柄菌发育的局部集体阈值，以促进细胞间通讯和多细胞形成。 DPF 高表达区域成为细胞间信号响应、多细胞形成和细胞命运决定的中心。此外，DPF 具有独立的细胞自主功能，用于调节细胞粘附和生长。 心肌功能障碍通常与心脏脂滴（LD）的积累有关。 Perilipin 2 (Plin2) 是一种 LD 蛋白，参与细胞内 LD 的形成、稳定性和运输活动。尽管 Plin2 在心脏中高表达，但人们对其在心肌脂质储存中的作用知之甚少。最近的一份报告显示，Plin2 的心脏过度表达会导致大量心肌脂肪变性，表明 Plin2 可以稳定 LD。在这项研究中，我们假设 Plin2 缺陷会导致心肌脂质储存减少。与我们的假设相反，我们发现 Plin2-/- 小鼠心脏中甘油三酯的积累增加，特别是心肌细胞中。尽管Plin2-/-小鼠心脏中的脂质水平显着升高，但它们在基线条件和轻度应激下具有正常的心脏功能。然而，在诱导心肌梗塞后，与 Plin2+/+ 小鼠相比，Plin2-/- 小鼠的每搏输出量和心输出量减少。我们进一步证明，Plin2 缺陷心脏中甘油三酯积累的增加是由脂肪自噬改变引起的。总之，我们的数据表明 Plin2 对于 LD 的正确水解非常重要。 米色脂肪细胞可以以热量的形式耗散能量。代谢和基因表达之间的精细沟通对于米色脂肪细胞的调节非常重要。尽管脂滴 (LD) 结合蛋白在脂肪组织生物学中发挥重要作用，但 perilipin 3 (Plin3) 是否参与米色脂肪细胞形成和产热活动的调节仍不清楚。在这项研究中，我们证明 Plin3 消融会刺激腹股沟白色脂肪组织 (iWAT) 中的米色脂肪细胞和产热基因表达。与野生型小鼠相比，Plin3 敲除小鼠具有耐冷性，并且 iWAT 中的基础脂肪分解和刺激脂肪分解增强，诱导过氧化物酶体增殖物激活受体 (PPAR) 激活。在脂肪细胞中，Plin3 缺陷促进 PPAR 靶基因和解偶联蛋白 1 的表达以及冷刺激下多房 LD 的形成。此外，成纤维细胞生长因子21的表达和分泌上调，这归因于Plin3缺陷的脂肪细胞中PPAR的激活。这些数据表明，Plin3 作为一种内在保护因子，通过限制脂质代谢和生热基因表达来防止无效的米色脂肪细胞形成。