SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 10919481
• 负责人: ALAN R KIMMEL
• 金额: $ 155.05万
• 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/10919481
• 关键词: Adenylate Cyclase; Adhesions; Adipose tissue; Affect; Affinity; Amino Acids; Anabolism; Binding; Binding Proteins; Biochemical; Body Weight; Calcium; Calpain; Cardiovascular Diseases; Catalogs; Cell Communication; Cell Density; Cell Survival; Cell physiology; Cells; Characteristics; Chemotactic Factors; Chemotaxis; Cholesterol; Chromatin; Circulation; Citric Acid Cycle; Communication; Complex; Coupled; Cues; Cyclic AMP; Cytoplasm; Cytoplasmic Tail; Data; Derivation procedure; Development; Dictyostelium; Dioxygenases; Ensure; Epigenetic Process; Epitopes; Eukaryota; Eukaryotic Cell; Family; Fasting; Fatty Liver; Feedback; Folic Acid; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Genomics; Genotype; Glucose; Glycolysis; Growth; Growth and Development function; Hepatic; Heterodimerization; High Fat Diet; Homeostasis; Homo; Hydrolysis; Image; Individual; Insulin Resistance; Integral Membrane Protein; Kinetics; Life Cycle Stages; Ligands; Link; Lipid Mobilization; Lipids; Liver; Mammals; Maps; Membrane; Messenger RNA; Metabolic; Metabolic Diseases; Metabolic Pathway; Minor; Modeling; Molecular; Mouse Strains; Movement; Mus; Natural Immunity; Nonesterified Fatty Acids; Nutrient; Nutrient Depletion; Obesity; Organelles; Organism; Pathway interactions; Pattern; Pentosephosphate Pathway; Peptide Hydrolases; Periodicity; Permeability; Phagocytes; Phosphotransferases; Play; Population; Process; Prokaryotic Cells; Property; Protein Family; Protein Secretion; Proteins; RNA Splicing; Receptor Signaling; Regulation; Research; Risk Factors; Role; Signal Pathway; Signal Transduction; Sirolimus; Starvation; Succinates; Surface; System; Territoriality; Testing; Time; Tissues; Totipotent cell; Transcription Process; Transmembrane Domain; United States National Institutes of Health; Variant; Withdrawal; alpha ketoglutarate; cell growth; cell motility; chromatin modification; cofactor; density; desensitization; diet-induced obesity; directional cell; embryonic stem cell; experimental study; glucose tolerance; human disease; interest; mammalian genome; member; metabolic abnormality assessment; metabolic phenotype; metabolomics; molecular modeling; mouse model; novel; perilipin; perilipin A; phosphoric diester hydrolase; pluripotency; posttranscriptional; preference; receptor; recruit; response; self organization; steroid hormone; trafficking; transcriptome; transcriptome sequencing; vector

Modeling cAMP Oscilations - Self-organized and excitable signaling activities play important roles in a wide range of cellular functions in eukaryotic and prokaryotic cells. Cells require signaling networks to communicate amongst themselves, but also for response to environmental cues. Such signals involve complex spatial and temporal loops that may propagate as oscillations or waves. 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 cAMP synthesis/degradation and directional cell movement, that oscillate at 6 min intervals. Developmental cAMP oscillations are characterized by a rise in cAMP synthesis and accumulation, followed by cessation of cAMP synthesis and increased cAMP degradation, with the cycle repeating with a defined temporal periodicity. Although many immediate components that regulate cAMP signaling (including receptors, G proteins, adenyl cyclase, phosphodiesterases, and kinases) are known, others are only inferred. Using biochemical experiments coupled with gene inactivation studies, we have identified new component members and model an integrated large (>25), multi-component kinetic pathway involving activation, inactivation (adaptation), re-activation (re-sensitization), feed-forward, and feed-back controls to generate developmental cAMP oscillations. Metabolomics - Changes in nutrients affect diverse cellular networks, making it challenging to distinguish metabolic paths that regulate growth from a switch to development. The life cycle of Dictyostelium is an excellent model to study metabolic signatures. Dictyostelium grow as single cells in nutrient-rich media, but, with nutrient withdrawal, growth ceases and cells enter multi-cell development. We developed conditions for rapid cell growth in rich-media, but where rapamycin-targeted inactivation of mTORC1 leads to a growth-to-development fate switch. We have shown that nutrient (glucose, amino acids) withdrawal significantly reduces many intermediates within most metabolic pathways, thus, negatively impacting glycolysis, the TCA cycle, pentose-phosphate shunt, etc. Rapamycin-induced development in the absence of nutrient withdrawal is expected to have a more limited influence on metabolic pathways. As part of the trans-NIH Metabolomics Consortium, we have undertaken time-course analyses of metabolomic changes in response to starvation- and rapamycin-induced development, to identify metabolic changes that are associated with a growth-to-development transition, but that are independent of nutrient depletion. We wish to identify metabolic changes that result form development (e.g. autophagic products), but also regulatory metabolites that may promote (e.g. AMP) or inhibit development. Initial results indicate that >5000 metabolite concentration differences are seen between starved-developed and growing cells, whereas <500 differ between growth and rapamycin-induced development in the absence of nutrient withdrawal. We anticipate identifying a defined catalog of metabolites whose concentrations are highly varied during development, independent of nutrient withdrawal. Some may function as epigenetic regulators of cell-fate change. As an example, a-ketoglutarate is a co-factor for dioxygenases that suppress repressive chromatin modifications with impact to transcription and a-ketoglutarate/succinate TCA component ratio differences can promote or suppress ES cell pluripotency. Our preliminary data in Dictyostelium indicate a >4-fold decrease in relative a ketoglutarate to succinate levels as development proceeds. We have further demonstrated that methyl-derivates of a ketoglutarate, for enhanced cell permeability, completely block developmental induction, even under starved conditions, without an effect on cell viability and growth. Data suggest that a ketoglutarate concentration is the primary effector, as developmental inhibition cannot be reversed with increased levels of succinate moieties. We will look for changes in the transcriptome and in chromatin organization that are specific to a-ketoglutarate treatment. Comparison of RNAseq data from cells starved in the presence or absence of exogenous a-ketoglutarate may further discriminate transcriptional changes closely associated with development from those only responsive to nutrient withdrawal. Growth-to-Development Developmental aggregation in Dictyostelium is lost at low cell density, but aggregation at non-permissive cell densities is rescued with secreted factor DPF1. Secreted DPF1 is synthesized as a larger precursor, single-pass transmembrane protein that is released by proteolytic cleavage and ectodomain shedding, leaving a 10 kDa transmembrane (TM) fragment. The TM/cytoplasmic domain of DPF1 possesses independent, cell autonomous activity for cell-substratum adhesion and cellular growth. We have created vectors that solely express the secreted or TM/cytoplasmic forms to understand the different functions. We have also identified a new gene DPF2, which is closely linked to DPF1, that encodes a sequence related protein with similar processing and ectodomain shedding properties. Ectodomain cleavage of both DPF1 and DPF2 is largely dependent upon calcium and calcium-dependent proteases (calpains). Secreted p150 kDa fragments of DPF1 and DPF2 have been purified in mg quantities and are being analyzed by MS/MS to map the specific cleavage sequences. We hypothesize there is pathway interaction between DPF1 and DPF2 and are testing this directly in mixing experiments with differentially epitope-tagged versions of DPF1 and DPF2. We have shown homo- and hetero-dimerization of the transmembrane domains of both DPF1 and DPF2. Lipid Storage during Fasting- 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. Plin2 binds hepatic LDs with expression levels that correlate with TAG content. We investigated the role of Plin2 in hepatic LD storage in fed and fasted plin2+/+ and plin2-/- mice. Chow-fed plin2-/- mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels to WT. Overnight fasting stimulates the degradation of stored adipose TAGs, with release of non-esterified fatty acids for circulation. Fasted plin2-/- mice showed substantially reduced accumulation of hepatic TAG compared to fasted WT. RNAseq revealed minor differences in hepatic gene expression between fed plin2+/+ and plin2-/- mice but marked differences in expression between fasted plin2+/+ and plin2-/- mice. Plin2 regulates hepatic lipid droplet size and accumulation of neutral lipid species in the fasted state. Hepatosteatosis - Recent studies describe transcriptome changes associated with hepatosteatosis, but it has been difficult to separate the effects on hepatic gene expression of fatty liver from that of obesity. We studied a plin2-/- mouse model, under conditions that are highly protective to hepatostaetosis, but not diet-induced obesity. We determined the mechanistic functions that protect plin2-/- livers from lipid accumulation and using RNAseq, compared hepatic transcriptomes of chow-fed or high-fat diet plin2+/+ and plin2 /- mice. We show that the Plin2 genotype, and accordingly hepatosteatosis, has a more limited impact on hepatic gene expression than does diet-induced obesity.

模拟 cAMP 振荡 - 自组织和可兴奋的信号传导活动在真核和原核细胞的多种细胞功能中发挥着重要作用。细胞需要信号网络在它们之间进行通信，也需要对环境线索做出反应。这些信号涉及复杂的空间和时间环路，可以作为振荡或波传播。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。发育期 cAMP 振荡的特征是 cAMP 合成和积累增加，随后 cAMP 合成停止和 cAMP 降解增加，该循环以定义的时间周期重复。尽管调节 cAMP 信号传导的许多直接成分（包括受体、G 蛋白、腺苷酸环化酶、磷酸二酯酶和激酶）是已知的，但其他成分仅是推测的。利用生化实验与基因失活研究相结合，我们已经确定了新的成分成员，并建立了一个集成的大型（>25）多成分动力学途径模型，涉及激活、失活（适应）、重新激活（重新敏化）、前馈和反馈控制以产生发育性 cAMP 振荡。 代谢组学 - 营养物质的变化影响不同的细胞网络，因此很难区分调节生长从转变到发育的代谢路径。盘基网柄菌的生命周期是研究代谢特征的绝佳模型。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。我们开发了细胞在丰富培养基中快速生长的条件，但雷帕霉素靶向的 mTORC1 失活会导致生长到发育的命运转换。我们已经表明，营养物质（葡萄糖、氨基酸）的撤除会显着减少大多数代谢途径中的许多中间体，从而对糖酵解、TCA 循环、戊糖-磷酸分流等产生负面影响。预计在没有营养撤退的情况下雷帕霉素会诱导发育对代谢途径的影响更为有限。作为跨 NIH 代谢组学联盟的一部分，我们对饥饿和雷帕霉素诱导的发育产生的代谢组变化进行了时间过程分析，以确定与生长到发育转变相关的代谢变化，但这些变化是与营养消耗无关。我们希望识别发育引起的代谢变化（例如自噬产物），以及可能促进（例如 AMP）或抑制发育的调节代谢物。初步结果表明，在饥饿发育和生长的细胞之间观察到>5000的代谢物浓度差异，而在没有营养撤退的情况下，生长和雷帕霉素诱导的发育之间的代谢物浓度差异<500。我们预计会确定一个明确的代谢物目录，其浓度在发育过程中变化很大，与营养吸收无关。有些可能充当细胞命运变化的表观遗传调节剂。例如，α-酮戊二酸是双加氧酶的辅助因子，可抑制抑制性染色质修饰，从而影响转录，而α-酮戊二酸/琥珀酸 TCA 成分比例差异可以促进或抑制 ES 细胞的多能性。我们在盘基网柄菌属中的初步数据表明，随着发育的进行，酮戊二酸与琥珀酸的相对水平下降了 4 倍以上。我们进一步证明，酮戊二酸的甲基衍生物可以增强细胞通透性，甚至在饥饿条件下也能完全阻断发育诱导，而不影响细胞活力和生长。数据表明酮戊二酸浓度是主要效应器，因为发育抑制不能随着琥珀酸部分水平的增加而逆转。我们将寻找α-酮戊二酸治疗特有的转录组和染色质组织的变化。比较在存在或不存在外源α-酮戊二酸的情况下饥饿细胞的RNAseq数据可以进一步区分与发育密切相关的转录变化和仅对营养缺乏有反应的细胞。 生长发育 盘基网柄菌的发育聚集在低细胞密度下会消失，但在不允许的细胞密度下聚集会被分泌因子 DPF1 拯救。分泌型 DPF1 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释放，留下 10 kDa 跨膜 (TM) 片段。 DPF1 的 TM/细胞质结构域对于细胞-基质粘附和细胞生长具有独立的细胞自主活性。我们创建了仅表达分泌形式或 TM/细胞质形式的载体，以了解不同的功能。我们还发现了一个新基因 DPF2，它与 DPF1 密切相关，编码具有相似加工和胞外域脱落特性的序列相关蛋白。 DPF1 和 DPF2 的胞外域裂解很大程度上依赖于钙和钙依赖性蛋白酶（钙蛋白酶）。 DPF1 和 DPF2 分泌的 p150 kDa 片段已纯化至毫克级，并通过 MS/MS 进行分析以绘制特定切割序列。我们假设 DPF1 和 DPF2 之间存在通路相互作用，并在使用差异表位标记版本的 DPF1 和 DPF2 的混合实验中直接测试这一点。我们已经展示了 DPF1 和 DPF2 跨膜结构域的同源二聚化和异源二聚化。 禁食期间的脂质储存——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，可储存细胞能量、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。 Plin2 结合肝脏 LD，其表达水平与 TAG 含量相关。我们研究了 Plin2 在进食和禁食的 plin2+/+ 和 plin2-/- 小鼠肝脏 LD 储存中的作用。食物喂养的 plin2-/- 小鼠的体重、代谢表型、葡萄糖耐量以及循环 TAG 和总胆固醇水平与 WT 相当。隔夜禁食会刺激储存的脂肪标签的降解，释放非酯化脂肪酸用于循环。与禁食的WT小鼠相比，禁食的plin2-/-小鼠表现出肝脏TAG积累显着减少。 RNAseq揭示了进食的plin2+/+和plin2-/-小鼠之间肝脏基因表达的微小差异，但禁食的plin2+/+和plin2-/-小鼠之间的表达存在显着差异。 Plin2 在禁食状态下调节肝脏脂滴大小和中性脂质物质的积累。 肝脂肪变性 - 最近的研究描述了与肝脂肪变性相关的转录组变化，但很难区分脂肪肝和肥胖对肝脏基因表达的影响。我们研究了 plin2-/- 小鼠模型，该模型在对肝硬化具有高度保护作用的条件下进行，但对饮食诱导的肥胖没有作用。我们确定了保护 plin2-/- 肝脏免受脂质积累的机制功能，并使用 RNAseq 比较了食物喂养或高脂肪饮食的 plin2+/+ 和 plin2 /- 小鼠的肝脏转录组。我们发现，与饮食引起的肥胖相比，Plin2 基因型以及相应的肝脂肪变性对肝基因表达的影响更为有限。

项目成果

Oscillatory signaling and network responses during the development of Dictyostelium discoideum.

盘基网柄菌发育过程中的振荡信号和网络反应。

• DOI: 10.1016/j.arr.2008.04.003
• 发表时间: 2008
• 期刊: Ageing research reviews
• 影响因子: 13.1
• 作者: McMains,VanessaC;Liao,Xin-Hua;Kimmel,AlanR
• 通讯作者: Kimmel,AlanR

Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets.

• DOI: 10.1242/jcs.104943
• 发表时间: 2012-09-01
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Hsieh K;Lee YK;Londos C;Raaka BM;Dalen KT;Kimmel AR
• 通讯作者: Kimmel AR

Isolated Plin5-deficient cardiomyocytes store less lipid droplets than normal, but without increased sensitivity to hypoxia.

• DOI: 10.1016/j.bbalip.2020.158873
• 发表时间: 2020-12
• 期刊: Biochimica et biophysica acta. Molecular and cell biology of lipids
• 作者: Yuchuan Li;M. Torp;F. Norheim;P. Khanal;A. Kimmel;K. Stensløkken;J. Vaage;K. Dalen

Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium.

• DOI: 10.1101/gr.216309.116
• 发表时间: 2017-04
• 期刊: Genome research
• 影响因子: 7
• 作者: Platt JL;Kent NA;Kimmel AR;Harwood AJ
• 通讯作者: Harwood AJ

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development.

DPF是一种细胞密度传感因子，在盘基网柄菌生长发育过程中具有细胞自主和非自主功能。

• DOI: 10.1186/s12915-019-0714-9
• 发表时间: 2019
• 期刊: BMC biology
• 影响因子: 5.4
• 作者: Meena,NetraPal;Jaiswal,Pundrik;Chang,Fu-Sheng;Brzostowski,Joseph;Kimmel,AlanR
• 通讯作者: Kimmel,AlanR