SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 10259345
• 负责人: ALAN R KIMMEL
• 金额: $ 149.67万
• 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/10259345
• 关键词: Acute; Adhesions; Adipose tissue; Adrenal Cortex; Adrenal Glands; Adult; Affinity; Age; Agonist; Anabolism; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Brain Hypoxia-Ischemia; Carbon Dioxide; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Aggregation; Cell Communication; Cell Density; Cell physiology; Cells; Ceroid; Characteristics; Chemotactic Factors; Chemotaxis; Cholesterol; Cholesterol Esters; Complex; Corticosterone; Corticotropin; Culture Media; Cysteine; Cytoplasmic Tail; Data; Development; Developmental Process; Dictyostelium; Discrimination; Equilibrium; Eukaryota; Excision; Exhibits; Extracellular Protein; FRAP1 gene; Family; Family member; Fatty Acids; Fatty Liver; Food; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic; Glucose; Glycogen; Growth; Growth and Development function; Hamartoma; Heart; Human; Hydrolysis; Hypoxia; Individual; Infarction; Insulin Resistance; Integral Membrane Protein; Ischemia; Knockout Mice; Length; Ligands; Lipase; Lipids; Lipolysis; Mammalian Cell; Mammals; Mediating; Membrane; Messenger RNA; Metabolic; Metabolic Diseases; Mitochondria; Modeling; Molecular Genetics; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Mus; Muscle Fibers; Mutation; Myocardial; Natural Immunity; Nutrient; Nutrient Depletion; Oleic Acids; Organelles; Organism; Oxides; Pathway interactions; Phagocytes; Phagocytosis; Pharmaceutical Preparations; Pharmacology; Phenocopy; Phenotype; Phosphatidylglycerols; Phospholipids; Physiologic intraventricular pressure; Physiological; Play; Positioning Attribute; Proliferating; Protein Sequence Analysis; Proteins; RNA; RNA-Binding Proteins; Receptor Signaling; Regulation; Regulatory Pathway; Regulon; Risk Factors; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sirolimus; Source; Specificity; Starvation; Structure; Surface; TIS11 protein; Techniques; Transcript; Transcription Process; Triglycerides; Weight; Withdrawal; Zinc Fingers; alpha-tocopherol transfer protein; amplification detection; cell growth; density; energy balance; experimental study; extracellular; fMet-Leu-Phe receptor; fatty acid oxidation; glycogen metabolism; human disease; improved; loss of function; mRNA Expression; macrophage; mitochondrial membrane; molecular modeling; mutant; non-oncogenic; novel; null mutation; overexpression; oxidation; perilipin; perilipin A; prevent; programs; response; reverse cholesterol transport; sensor; steroid hormone; tumorigenesis; uptake

Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation; mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We showed functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments indicate novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions. Cellular functions can be regulated by cell-cell interactions and influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources. As nutrients become depleted, they initiate a multi-cell developmental program dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor. We show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth. Post-transcriptional processes mediated by mRNA binding proteins represent important control points in the regulation of gene expression. In mammals, mRNAs containing specific AU-rich motifs are regulated by proteins of the tristetraprolin (TTP) family, which bind to these motifs through a tandem zinc finger (TZF) domain. This binding leads to promotion of subsequent deadenylation and decay, partly through a conserved carboxyl-terminal CNOT1 binding domain. Four TTP family members are expressed in mice, and possible functional redundancy makes structure-function explorations difficult. We explored the physiological role of the single TTP family member (ttpA) expressed in Dictoystelium discoideum. Two null mutants exhibited slow growth and a multinuclear phenotype. A cysteine point mutant within the TZF domain, which should completely prevent mRNA binding, phenocopied the two null mutations exactly in terms of gene expression patterns. The carboxyl-terminal mutation, which removed the predicted CNOT1 binding domain, exhibited a phenotype indistinguishable from that of the null mutants, raising the possibility that this domain is primarily responsible for interactions with the CCR4/NOT mRNA decay apparatus in this species. Three transcripts which are highly upregulated in the loss-of-function mutants, are co-regulated during differentiation and possess the conserved AU-target motif and. These data suggest that ttpA controls an RNA "regulon", in which a single RNA binding protein co-regulates the expression of a gene group. Most cells store triacylglycerol (TAG) in cytoplasmic lipid droplets (LDs), but steroidogenic cells store primarily cholesteryl esters (CEs). The LD binding protein Plin2 regulates degradation of LDs containing TAG. To determine if Plin2 is also regulates CEs, we characterized adrenals of Plin2 null mice. We observed significantly enlarged adrenal glands, increased size and numbers of CE-LD, elevated cellular free cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport in adrenals of Plin2-/- mice as compared to Plin2+/+ mice. Mobilization of CE-LDs and secretion of corticosterone induced by ACTH stimulation or starvation were unaffected by the lack of Plin2 in young mice. There was also accumulation of ceroid-like structures containing multilamellar bodies in the adrenal cortex-medulla boundary accompanied with high levels of adrenal phosphatidylglycerols, implying that these lipids are components of the accumulated ceroid-like structures. Our findings indicate that Plin2 is important for regulation of CE-LDs and cellular cholesterol balance in adrenal cortical cells. Perilipin 5 (Plin5) is abundantly expressed in heart where it binds to cardiac lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5-/- mice to study the role of Plin5 for tolerance to hypoxia and ischemia, fatty acid uptake, LD accumulation, and fatty acid oxidation. Cardiomyocytes from Plin5-/- mice stored less LDs than Plin5+/+ following oleic acid stimulation, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/- cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia. Deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia in isolated, perfused hearts. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight and elevated Myh7 and Cd36 mRNA expression compared to Plin5+/+. Still, most other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by age or removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content does not appear to be of vital importance for tolerance to acute hypoxia and ischemia.

多蛋白复合物 mTORC1 和 mTORC2 是真核生物生长和发育所必需的。 mTORC1是一种营养传感器，整合代谢信号和能量状态来调节细胞生长/增殖； mTORC2 主要调节发育过程。盘基网柄菌在丰富的生长培养基中增殖，但在营养耗尽时开始发育。两种 mTOR 复合物在盘基网柄菌中都发挥着重要作用，盘基网柄菌的生长和发育周期分别独立地需要 mTORC1 或 mTORC2。盘基网柄菌中 mTORC1 和 mTORC2 的许多蛋白质关联和调控途径与哺乳动物细胞具有相似性，并且对免疫抑制药物雷帕霉素的抑制具有特异性。在盘基网柄菌中，mTORC1 功能在饥饿诱导发育时失活，但即使在没有营养撤退的情况下，发育也是通过雷帕霉素介导的 mTORC1 活性抑制直接诱导的。在不损失营养的情况下，通过药理抑制 mTORC1，可以鉴定出一类重要的上调、发育相关的信号基因和下调的生长基因。我们展示了整合 mTORC1/mTORC2 活性的功能通路调节，并强调了 mTORC2 活性的小 GTPase 调节的复杂性。最后，上位实验表明盘基网柄菌中新的上游通路串扰需要 mTORC1 和 mTORC2，但用于单独且独立的下游功能。 细胞功能可以通过细胞间相互作用进行调节，并受到细胞外、密度依赖性信号传导因子的影响。盘基网柄菌在营养丰富的来源中以单个细胞的形式生长。当营养物质耗尽时，它们会启动依赖于细胞密度阈值的多细胞发育程序。我们假设新型分泌蛋白可能作为密度敏感因子，在特定的细胞密度阈值下促进多细胞发育命运决定，并使用盘基网柄菌来鉴定此类因子。我们发现，盘基网柄菌属中的多细胞发育聚集在局部细胞密度最小（2倍）减少时丧失。值得注意的是，通过添加来自高密度、发育活性细胞的条件培养基，可以挽救非允许细胞密度下的发育聚集反应。使用低密度细胞的挽救聚集作为分析，我们纯化了具有密度聚集活性的单一 150 kDa 细胞外蛋白。 MS/MS 肽序列分析确定了基因序列，并且过表达全长蛋白的细胞比亲代细胞积累了更高水平的发育促进因子 (DPF) 活性，从而使细胞能够以较低的细胞密度聚集；与 WT 相比，缺乏该 DPF 基因的细胞缺乏密度依赖性发育聚集活性，并且需要更高的细胞密度来进行细胞聚集。密度聚集活性与标记版本的 DPF 共同纯化，并且标记亲和纯化的 DPF 具有密度聚集活性。在与 WT 的混合发育中，过表达 DPF 的细胞优先定位于多细胞聚集的中心，并在细胞分化过程中定义细胞命运的选择。最后，我们证明 DPF 被合成为更大的前体、单次跨膜蛋白，其中 p150 片段通过蛋白水解切割和胞外域脱落而释放。 DPF 的 TM/细胞质结构域具有细胞-基质粘附和细胞生长的细胞自主活性。 由 mRNA 结合蛋白介导的转录后过程是基因表达调节中的重要控制点。在哺乳动物中，含有特定富含 AU 基序的 mRNA 受到三四脯氨酸 (TTP) 家族蛋白的调节，该家族通过串联锌指 (TZF) 结构域与这些基序结合。这种结合部分通过保守的羧基末端 CNOT1 结合域促进随后的去腺苷化和衰变。四个 TTP 家族成员在小鼠中表达，可能的功能冗余使得结构功能探索变得困难。我们探讨了盘基网菌中表达的单个 TTP 家族成员 (ttpA) 的生理作用。两个无效突变体表现出生长缓慢和多核表型。 TZF 结构域内的半胱氨酸点突变体应该完全阻止 mRNA 结合，它在基因表达模式方面准确地复制了两个无效突变。羧基末端突变去除了预测的 CNOT1 结合结构域，表现出与无效突变体无法区分的表型，这提高了该结构域主要负责与该物种中 CCR4/NOT mRNA 衰变装置相互作用的可能性。三个转录物在功能丧失突变体中高度上调，在分化过程中共同调节，并具有保守的 AU 靶基序。这些数据表明ttpA控制RNA“调节子”，其中单个RNA结合蛋白共同调节基因组的表达。 大多数细胞在细胞质脂滴（LD）中储存三酰甘油（TAG），但类固醇生成细胞主要储存胆固醇酯（CE）。 LD 结合蛋白 Plin2 调节含有 TAG 的 LD 的降解。为了确定 Plin2 是否也调节 CE，我们对 Plin2 缺失小鼠的肾上腺进行了表征。我们观察到与 Plin2+/+ 小鼠相比，Plin2-/- 小鼠的肾上腺显着增大，CE-LD 的大小和数量增加，细胞游离胆固醇水平升高，巨噬细胞标记物和促进胆固醇反向转运的基因表达增加。在年轻小鼠中，ACTH 刺激或饥饿诱导的 CE-LD 的动员和皮质酮的分泌不受 Plin2 缺乏的影响。在肾上腺皮质-髓质边界处还积累了含有多层体的蜡样结构，并伴有高水平的肾上腺磷脂酰甘油，这表明这些脂质是积累的蜡样结构的组成部分。我们的研究结果表明，Plin2 对于调节肾上腺皮质细胞中的 CE-LD 和细胞胆固醇平衡很重要。 Perilipin 5 (Plin5) 在心脏中大量表达，与心脏脂滴 (LD) 结合，促进 LD 和线粒体之间的物理相互作用。我们从成年 Plin5+/+ 和 Plin5-/- 小鼠中分离出心肌细胞，以研究 Plin5 在耐缺氧和缺血、脂肪酸摄取、LD 积累和脂肪酸氧化方面的作用。油酸刺激后，Plin5-/- 小鼠的心肌细胞储存的 LD 低于 Plin5+/+，但当脂肪甘油三酯脂肪酶活性受到抑制时，其水平与 Plin5+/+ 心肌细胞相当。 Plin5+/+ 和 Plin5-/- 心肌细胞将脂肪酸氧化成 CO2 的能力相似，但 Plin5-/- 心肌细胞的细胞内脂肪酸氧化中间体短暂增加。用油酸预孵育后，Plin5-/-心肌细胞保留了较高含量的糖原，并表现出改善的缺氧耐受性。 Plin5 的缺失对离体灌注心脏缺血后的心室压力或梗塞面积没有重要影响。与 Plin5+/+ 相比，老 Plin5-/- 小鼠的心脏三酰甘油水平降低，心脏重量增加，Myh7 和 Cd36 mRNA 表达升高。尽管如此，大多数涉及脂肪酸氧化、糖原代谢和葡萄糖利用的其他基因基本上不会因年龄或 Plin5 的去除而发生变化。 Plin5 似乎主要通过抑制脂肪甘油三酯脂肪酶活性而不改变心脏脂肪酸氧化能力来促进心脏 LD 储存。 Plin5 的表达和心脏 LD 含量似乎对于急性缺氧和缺血的耐受性并不至关重要。