SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 10697814
• 负责人: ALAN R KIMMEL
• 金额: $ 143万
• 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/10697814
• 关键词: 3' Untranslated Regions; Acute; Adhesions; Adipocytes; Adipose tissue; Adrenal Cortex; Adrenal Glands; Adult; Affect; Agonist; Amino Acids; Anabolism; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Brain Hypoxia-Ischemia; Calcium; Calpain; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Communication; Cell Density; Cell physiology; Cells; Ceroid; Characteristics; Chemotactic Factors; Chemotaxis; Cholesterol; Cholesterol Esters; Complex; Corticosterone; Corticotropin; Cyclic AMP; Cytoplasmic Tail; Data; Development; Dictyostelium; Discrimination; Ensure; Epitopes; Equilibrium; Eukaryota; Family; Fatty Acids; Fatty Liver; Food; Gene Expression; Generations; Genes; Genetic Transcription; Genomics; Glucose; Glycogen; Growth; Growth and Development function; Heart; Homeostasis; Hydrolysis; Hypoxia; Image; Individual; Infarction; Insulin Resistance; Integral Membrane Protein; Ischemia; Kinetics; Life Cycle Stages; Ligands; Lipase; Lipid Mobilization; Lipids; Lipolysis; Mammalian Cell; Maryland; Mediating; Membrane; Messenger RNA; Metabolic; Metabolic Control; Metabolic Diseases; Metabolic Pathway; Mitochondria; Modeling; Molecular; Molecular Genetics; Molecular Target; Mouse Strains; Movement; Mus; Muscle Fibers; Mutation; Myocardial; Myocardial Ischemia; Natural Immunity; Null Lymphocytes; Nutrient; Nutrient Depletion; Oleic Acids; Organelles; Organism; Pathway interactions; Pattern; Peptide Hydrolases; Phagocytes; Phagocytosis; Phenotype; Phosphatidylglycerols; Phospholipids; Phosphotransferases; Physiologic intraventricular pressure; Population; Process; Property; Proteins; Receptor Signaling; Regulation; Research; Risk Factors; Role; Signal Pathway; Signal Transduction; Sirolimus; Source; Starvation; Structure; Succinates; Surface; System; TIS11 protein; Territoriality; Testing; Time; Tissues; Totipotent cell; Transcript; Transcription Process; Triglycerides; United States National Institutes of Health; Universities; Untranslated Regions; Water; Withdrawal; Zinc Fingers; alpha ketoglutarate; amplification detection; cell growth; cell motility; cell type; density; directional cell; energy balance; experimental study; fMet-Leu-Phe receptor; fatty acid oxidation; human disease; improved; interest; mRNA Decay; mRNA Stability; macrophage; medical schools; metabolic abnormality assessment; metabolomics; mitochondrial membrane; molecular modeling; mouse model; mutant; novel; overexpression; oxidation; perilipin; perilipin A; recruit; response; reverse cholesterol transport; sensor; single-cell RNA sequencing; steroid hormone; transcriptome sequencing; uptake; vector

Metabolomics - Changes in nutrient levels affect diverse cellular networks, making it challenging to distinguish metabolic paths that regulate growth or a switch to development. The life cycle of Dictyostelium presents an excellent model to such study metabolic signatures. Dictyostelium grow as single cells in nutrient-rich media, but, with nutrient withdrawal, growth ceases and cells enter multi-cell development. Kinases mTORC1 and AMPK are reciprocal energy sensors, to control cellular growth; mTORC1 is required for cell growth, activated by nutrients, and inactivated upon nutrient withdrawal. AMPK has opposite kinetics. In addition, mTORC1 and AMPK are negative regulators of each other, reinforcing reciprocal patterns. While both respond to specific metabolites (e.g. amino acids, AMP) they also control metabolic pathways. We have shown that rapamycin-targeted inactivation of mTORC1, in the absence of nutrient withdrawal, leads to reciprocal activation of AMPK, which is sufficient to effect a growth-to-developmental fate switch and to induce multi-cell development of Dictyostelium. Using an RNA-sequencing approach, we then identified mTORC1/AMPK-regulated transcriptional networks and associated signaling pathways that are essential for early developmental induction, but that are regulated independently of nutrient withdrawal. We use this system as part of The trans-NIH Metabolomics Consortium to identify metabolic changes that are associated with a growth-to-development transition, but that are not dependent of nutrient depletion. Preliminary data indicate a >4-fold increase in relative succinate to alpha ketoglutarate levels, as cells enter development and treatment with exogenous alpha ketoglutarate blocks development; molecular targets are being investigated. We also collaborate with Dr. Vivek Garg at the University of Maryland School of Medicine on related mitochondrial Ca+2 regulatory modes. Growth-to-Development Fate Decisions - When Dictyostelium are starved for nutrients, cells within a territorial space secrete cAMP. Proximal cells move inward toward cAMP and relay the cAMP outward to recruit additional cells. To ensure directed inward movement, cells go through adapted and de-adapted states, for both cAMP synthesis/degradation and directional cell movement, that oscillate at 6 min intervals. Such cell-cell interaction responses are enhanced by increased cell density. We had identified a secreted 150 kDa protein, termed DPF (Development Promoting Factor), which possesses density dependent activity. Cells that overexpress DPF develop at low cell densities; cells deficient for DPF require higher cell density, compared to WT. DPF is synthesized as a larger precursor, single-pass transmembrane protein that is released by proteolytic cleavage and ectodomain shedding. The secreted 150 kDa factor is sufficient to regulate development non-autonomously, but the TM/cytoplasmic domain of DPF possesses independent cell autonomous activity for cell-substratum adhesion and cellular growth. We have created vectors that express only the secreted or TM/cytoplasmic forms to test mechanisms. We have also identified a sequence related protein with similar DPF properties and potential pathway. The phenotypes of individual-null cells are not identical but are consistent with a model where interactions mediate cell density sensing and development. Such a model is being tested directly in cell mixing experiments with WT and mutant proteins. We have also shown that DPF ectodomain cleavage is largely inhibited by depletion of calcium and by mutation of calcium-dependent proteases (calpains). The cleaved factor has been purified and is being analyzed by MS/MS to identify the cleavage sequence. To understand the non-autonomous effects of DPF on heterologous cell gene expression, we have prepared single-cell RNAseq time courses, comparing 100% DPF-nulls to 99% null/1% over-expressor mixes. SC RNAseq we enable us to unequivocally identify cells that do not express endogenous DPF and to determine how they specifically respond to exogenously added factor. Regulation of mRNA Stability - Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. We have identified a tandem zinc finger protein TTP in Dictyostelium that promotes mRNA decay, likely through interaction with CCR4-NOT protein complexes. Six mRNA transcripts are consistently upregulated in null and point mutants compared to WT, and the 3'-untranslated regions (3'-UTRs) of all six contain multiple, overlapping UUAUUUAUU motifs; one 3'-UTR conferred TTP post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mRNA mutations in the core UA-rich motifs. Expression of the Dictyostelium protein in mammalian cells promotes mRNA decay of mammalian transcripts that contain these motifs, despite evolutionary divergence of nearly a billion years. Function requires more than a single motif. Dependent may also require open structures within the 3UTRs. Interactions with mRNAs and proteins are being followed with IP of functionally active epitope tagged TTP. Regulation of Lipid Storage - Excessive cellular lipid storage can be a risk factor for metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. The perilipins are a multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis. Perlipin 5 (Plin5) is abundantly expressed in the heart where it binds to lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We studied fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia in isolated cardiomyocytes from adult Plin5+/+ and Plin5-/-. Plin5-/- cardiomyocytes stored less LDs than did Plin5+/+, which was reversed by inhibition of the adipocyte triglyceride lipase (ATGL). Pre-incubation of Plin5-/- cardiomyocytes with oleic acids, increased glycogen and improved tolerance to hypoxia, by normalizing ventricular pressure and infarct size following ischemia. 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 in isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in actual mouse models during myocardial ischemia, emphasizing their more complex metabolic. Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in LDs, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs had been unclear. We showed that adrenal glands of Plin2-/- mice had significantly increased mass of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. They also had high levels of phosphatidylglycerols, which paralleled the accumulation of ceroid-like structures; secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were, nonetheless, similar in Plin2+/+ and Plin2-/- mice. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex.

代谢组学 - 营养水平的变化影响不同的细胞网络，使得区分调节生长或发育转变的代谢路径变得具有挑战性。盘基网柄菌的生命周期为此类研究代谢特征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，随着营养的减少，生长停止并且细胞进入多细胞发育。激酶 mTORC1 和 AMPK 是相互的能量传感器，用于控制细胞生长； mTORC1 是细胞生长所必需的，由营养物质激活，并在营养物质撤除后失活。 AMPK 具有相反的动力学。此外，mTORC1 和 AMPK 互为负调节因子，增强了相互模式。虽然两者都对特定代谢物（例如氨基酸、AMP）做出反应，但它们也控制代谢途径。我们已经证明，在没有营养撤退的情况下，雷帕霉素靶向的 mTORC1 失活会导致 AMPK 的相互激活，这足以影响生长到发育命运的转换并诱导盘基网柄菌的多细胞发育。然后，我们使用 RNA 测序方法确定了 mTORC1/AMPK 调节的转录网络和相关信号通路，这些通路对于早期发育诱导至关重要，但其调节独立于营养吸收。我们使用该系统作为跨 NIH 代谢组学联盟的一部分来识别与生长到发育过渡相关但不依赖于营养消耗的代谢变化。初步数据表明，随着细胞进入发育阶段，并且用外源性 α 酮戊二酸治疗会阻碍发育，琥珀酸与 α 酮戊二酸的相对水平增加了 4 倍以上；分子靶点正在研究中。我们还与马里兰大学医学院的 Vivek Garg 博士合作研究相关线粒体 Ca+2 调节模式。 生长发育命运决定 - 当盘基网柄菌缺乏营养时，领地空间内的细胞会分泌 cAMP。近端细胞向内移向 cAMP，并将 cAMP 向外传递以招募更多细胞。为了确保定向向内运动，细胞会经历适应和去适应状态，以进行 cAMP 合成/降解和定向细胞运动，以 6 分钟的间隔振荡。这种细胞间相互作用反应通过细胞密度的增加而增强。我们已经鉴定出一种分泌的 150 kDa 蛋白质，称为 DPF（发育促进因子），它具有密度依赖性活性。过度表达 DPF 的细胞在低细胞密度下发育；与 WT 相比，DPF 缺陷的细胞需要更高的细胞密度。 DPF 被合成为较大的前体、单次跨膜蛋白，通过蛋白水解切割和胞外域脱落而释放。分泌的 150 kDa 因子足以非自主调节发育，但 DPF 的 TM/细胞质结构域具有独立的细胞自主活性，用于细胞-基质粘附和细胞生长。我们创建了仅表达分泌形式或 TM/细胞质形式的载体来测试机制。我们还鉴定了一种具有相似 DPF 特性和潜在途径的序列相关蛋白。单个无效细胞的表型并不相同，但与相互作用介导细胞密度感知和发育的模型一致。这种模型正在使用 WT 和突变蛋白的细胞混合实验中直接进行测试。我们还表明，DPF 胞外域裂解在很大程度上受到钙消耗和钙依赖性蛋白酶（钙蛋白酶）突变的抑制。切割的因子已被纯化，并通过 MS/MS 进行分析，以鉴定切割序列。为了了解 DPF 对异源细胞基因表达的非自主影响，我们准备了单细胞 RNAseq 时间进程，比较 100% DPF 无效与 99% 无效/1% 过度表达混合。 SC RNAseq 使我们能够明确识别不表达内源 DPF 的细胞，并确定它们如何对外源添加因子做出特异性反应。 mRNA 稳定性的调节 - 由 mRNA 结合蛋白介导的转录后过程代表了基因表达中的重要控制点。我们在盘基网柄菌中发现了一种串联锌指蛋白 TTP，它可能通过与 CCR4-NOT 蛋白复合物相互作用来促进 mRNA 衰减。与 WT 相比，6 个 mRNA 转录物在零突变体和点突变体中持续上调，并且所有 6 个转录物的 3'-非翻译区 (3'-UTR) 均包含多个重叠的 UUAUUUAUU 基序；一个 3'-UTR 赋予异源 mRNA TTP 转录后稳定性调节作用，该调节作用被富含 UA 的核心基序中的 mRNA 突变所消除。尽管存在近 10 亿年的进化分歧，但盘基网柄菌蛋白在哺乳动物细胞中的表达会促进包含这些基序的哺乳动物转录本的 mRNA 衰减。功能需要多个主题。家属可能还需要 3UTR 内的开放结构。通过功能活性表位标记的 TTP 的 IP 跟踪 mRNA 和蛋白质的相互作用。 脂质储存的调节 - 细胞脂质储存过多可能是代谢紊乱的危险因素，包括胰岛素抵抗、心血管疾病和肝脂肪变性。细胞内脂滴是独特的细胞器，储存细胞能量、膜生物合成、类固醇激素合成和信号传导的代谢前体。周脂蛋白是​​一个多蛋白家族，其靶向脂滴表面并调节脂质储存和水解。 Perlipin 5 (Plin5) 在心脏中大量表达，它与脂滴 (LD) 结合，促进 LD 和线粒体之间的物理相互作用。我们研究了来自成人 Plin5+/+ 和 Plin5-/- 的分离心肌细胞的脂肪酸摄取、LD 积累、脂肪酸氧化和缺氧耐受性。 Plin5-/- 心肌细胞储存的 LD 比 Plin5+/+ 少，这可以通过抑制脂肪细胞甘油三酯脂肪酶 (ATGL) 来逆转。用油酸预孵育 Plin5-/- 心肌细胞，通过使缺血后的心室压力和梗塞面积正常化，增加糖原并提高对缺氧的耐受性。 Plin5 似乎主要通过抑制脂肪甘油三酯脂肪酶活性来促进心脏 LD 储存，而不改变心脏脂肪酸氧化能力。离体心肌细胞中 Plin5 的表达和心脏 LD 含量对于急性缺氧和缺血的耐受性影响不大，这与 Plin5 在实际小鼠模型中心肌缺血期间的保护作用形成鲜明对比，强调了其更复杂的代谢。胆固醇酯（CE）是胆固醇的不溶于水的运输和储存形式。类固醇生成细胞主要在 LD 中储存 CE，而大多数哺乳动物细胞类型主要在 LD 中储存三酰甘油 (TAG)。 LD 结合 Plin2 与富含 CE 和 TAG 的 LD 结合，尽管已知 Plin2 可以调节富含 TAG 的 LD 的降解，但其在调节富含 CE 的 LD 中的作用尚不清楚。我们发现 Plin2-/- 小鼠的肾上腺皮质细胞中富含 CE 的 LD 质量显着增加，细胞未酯化胆固醇水平升高，巨噬细胞标记物和促进胆固醇反向转运的基因表达增加。它们还具有高水平的磷脂酰甘油，这与类蜡质结构的积累是平行的。然而，Plin2+/+ 和 Plin2-/- 小鼠中促肾上腺皮质激素刺激或饥饿诱导的皮质酮分泌相似。我们的研究结果表明 Plin2 在调节肾上腺皮质中富含 CE 的 LD 和细胞胆固醇平衡方面发挥着重要作用。