Biosynthesis, Processing And Secretion Of Neuropeptides And Pituitary Hormones

神经肽和垂体激素的生物合成、加工和分泌

• 批准号: 8553819
• 负责人: Yoke p Loh
• 金额: $ 96.51万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/8553819
• 关键词: Actins; Acute; Adenylate Cyclase; Adrenergic Agents; Adrenergic Receptor; Adult; Affect; Age; Alzheimer' s Disease; Amino Acids; Anabolism; Animals; Anterior Pituitary Gland; Apoptosis; Area; Autocrine Communication; Binding; Biogenesis; Blood Pressure; Bone Density; Bone Marrow; C-terminal; Cardiac; Cell Line; Cell Nucleus; Cell membrane; Cells; Chromogranin A; Chronic stress; Cleaved cell; Clip; Coin; Collaborations; Complex; Corticotropin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Tail; Defect; Dense Core Vesicle; Development; Dexamethasone; Diabetes Mellitus; Disease; Ear; Electron Microscopy; Embryo; Embryonic Development; Endocrine; Endocrine system; Enzymes; Exocytosis; Fluorescence; Fluorescence Microscopy; Generations; Genes; Genetic Transcription; Genotype; Glucocorticoids; Glutamates; Golgi Apparatus; Heart Rate; Hippocampus (Brain); Hormones; Hydrogen Peroxide; Hypothalamic structure; In Situ Hybridization; Knockout Mice; Laboratories; Lead; Learning; Long-Term Potentiation; Measurement; Mediating; Mediator of activation protein; Membrane; Memory; Messenger RNA; Modeling; Mus; Mutation; Myocardium; N-terminal; Nerve Degeneration; Nervous System Physiology; Nervous system structure; Neurodegenerative Disorders; Neuroendocrine Cell; Neurons; Neuropeptides; Obesity; Organelles; Osteogenesis; Oxidative Stress; PC12 Cells; Pathway interactions; Peptide Hydrolases; Peptides; Physiological; Pituitary Gland; Pituitary Hormones; Play; Pro-Opiomelanocortin; Process; Proprotein Convertase 1; Proprotein Convertase 2; Prosencephalon; Protease Inhibitor; Proteins; Pyroglutamate; RNA Interference; Rattus; Recombinants; Role; Secretory Vesicles; Site; Slice; Small Interfering RNA; Somites; Sorting - Cell Movement; Sp1 Transcription Factor; Stem cells; Stress; Susceptibility Gene; Synapses; Synaptic Vesicles; Synaptophysin; Tail; Tissues; Transport Vesicles; Universities; Vesicle; Weaning; Wild Type Mouse; adrenergic; autocrine; carboxypeptidase H; cytotoxicity; falls; heart cell; knock-down; knockout animal; mRNA Expression; maternal separation; neurodevelopment; neuron loss; neuronal survival; neuroprotection; neurotrophic factor; nexin; novel; overexpression; peptide hormone; postnatal; prohormone; protein degradation; receptor; response; secretogranin III; synaptogenesis; trafficking

We have investigated the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway(RSP). Using our CPE knockout (KO) mouse, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII), which has been shown to bind POMC. SgIII, is found in neuroendocrine cells and is involved in trafficking of chromogranin A (CgA) to the RSP. We used RNA interference (siRNA) to knock down SgIII and CPE expression in AtT20 cells, we demonstrated increased POMC secretion via the constitutive secretory pathway in both cases. Increased constitutive secretion of CgA was only observed in the SgIII knockdown cells. In double CPE-SgIII knock down cells, increased constitutive secretion of POMC was observed and stimulated secretion of ACTH was perturbed. These results demonstrate that CPE is involved in the trafficking of POMC to the RSP; and that SgIII may play a compensatory role for CPE in the sorting of POMC to the RSP in addition to a more general role in the RSP trafficking process. We recently found that transmembrane CPE is not only associated with large dense core vesicles (LDCVs), but also with synaptic vesicles (SVs) in mouse hypothalamus and synaptic-like microvesicles in PC12 cells. High K+ stimulated release of glutamate from hypothalamic neurons was diminished in CPE-KO mice. Electron microscopy revealed that the number of SVs located in the pre-active zone (within 200nm of the plasma membrane at the active zone) of synapses was significantly decreased in hypothalamic neurons of CPE-KO mice compared with wild-type mice. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that overexpression of the CPE cytoplasmic tail reduced the steady-state level of synaptophysin-containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings show that the CPE cytoplasmic tail, which interacts with gamma adduccin and actin, is a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells. Our recent studies in pituitary AtT-20 cells have provided evidence for an autocrine mechanism for up-regulating LDCV biogenesis to replenish LDCVs following stimulated exocytosis of these vesicles. The autocrine signal was identified as serpinin, a novel 26 amino acid CgA-derived peptide cleaved from the C-terminal of CgA. Serpinin is released in an activity-dependent manner from LDCVs. Secreted serpinin was found to activate adenyl cyclase to increase cAMP levels, and protein kinase A in the cell. This then led to the translocation of the transcription factor sp1 from the cytoplasm into the nucleus and an increase in transcription of a protease inhibitor, protease nexin 1 (PN-1), which then inhibited granule protein degradation in the Golgi complex. The stabilization of those proteins increased their levels in the Golgi, resulting in significantly enhanced LDCV formation. We have also identified a N-terminal modified form of serpinin, pyroglutamate-serpinin (pGlu-serpinin) in pituitary AtT-20 cells and heart tissue. pGlu-serpinin was found to have neuroprotective activity against oxidative stress in AtT-20 cells and low K+-induced apoptosis in rat cortical neurons. In collaboration with Dr. Bruno Tota (University of Calabria), pGlu-serpinin was found to have positive inotropic activity in cardiac function, with no change in blood pressure and heart rate. pGlu-serpinin acts through a 1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway. pGlu-serpinin and other CgA derived cardioactive peptides emerge as novel -adrenergic inotropic and lusitropic modulators, Together, they can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation. CPE plays a significant role in obesity, and recently the gene has been coined an obesity susceptibility gene. We showed that extremely obese CPE-KO mice have low bone mineral density and concluded that that the lack of processing of pro-CART to mature CART, a peptide that promotes bone formation, is likely responsible for the poor bone density in these mice. Additionally in collaboration with Dr. Lecka-Czernik (Univ. of Toledo), we found that CPE is enriched in a rat messenchymal stem cell line from bone marrow and thus CPE may be involved in regulating bone formation at another level. This possibility is currently being explored. CPE-KO mice have deficiencies in their nervous system function, including learning and memory. We showed that in 6-14 week old CPE-KO mice, dendritic pruning was poor in cortical and hippocampal neurons which would affect synaptogenesis. Additionally electrophysiological measurements showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. This defect is attributed to the loss of neurons in the CA3 region of the hippocampus of CPE KO animals observed at 4 weeks of age and older. These neurons, which are normally enriched in CPE, were normal at 3 weeks of age just before the animals were weaned. When weaning was delayed a week, this degeneration was not observed till postnatal week 5 in the CPE KO mice. We have now shown that the degeneration is attributed to the stress of weaning which included maternal separation, ear tagging and tail clipping for genotyping. We also examined the effect of restrained stress on CPE expression in hippocampal neurons. When mice were subjected to acute restrained stress for 1h and then sacrificed 0, 1-24h post stress, they showed an immediate and transient decrease of CPE-mRNA expression in the hippocampus. In contrast after mild chronic stress (1h/day for 7days), which increases glucocorticoid secretion, the mice showed an increase in CPE mRNA and protein in the hippocampus, and no neuronal degeneration was evident. Furthermore, when hippocampal neurons were treated with synthetic glucocorticoid, dexamethasone, there was a significant increase in CPE mRNA and protein in the cells. These observations suggest that the increase in CPE may mediate neuronal survival during stress and lack of CPE results in neuronal degeneration. To this end, we have overexpressed or applied recombinant CPE exogenously in rat hippocampal neurons in culture and shown increased survival and neuroprotective effect of CPE on these neurons when subjected to oxidative stress with hydrogen peroxide treatment or glutamate cytotoxicity. The expression of CPE was examined in mouse embryos to determine if it could play a role in early embryonic development. We found that WT CPE and CPE-delta N mRNA was expressed as early as day E5.5 and increased each day, peaking at E8.5, falling slightly at E9.5 prior to expression of the endocrine system. CPE mRNA expression decreased sharply at E 10.5 -11.5 to below E5.5 levels and then increased sharply at E12.5 in parallel with the development of the endocrine system and continued to increase to adulthood. However, CPE-deltaN mRNA increased maximally at E10.5 followed by a precipitous decrease at E11.5-12.5, and then a small increase till PN1. In contrast to CPE, CPE-deltaN is absent in the adult hippocampus. In situ hybridization studies indicate that CPE and CPEdeltaN mRNA are expressed primarily in the fore brain and somites in mouse embryos. We have begun to study the role of CPE and CPE-delta N during embryonic development of the nervous system using neurospheres to study proliferation and differentiation.

我们已经研究了膜CPE和分泌蛋白III作为将POMC靶向受调节分泌途径（RSP）的分类受体的作用。使用我们的CPE敲除（KO）小鼠，我们表明50％的新合成POMC在垂体前叶的原代培养物中被降解，并表明由于缺乏CPE而没有有效分类RSP的颗粒，POMC的靶向是降级。但是，将其余的POMC分类到RSP中。补偿性分类受体的候选者是秘密素蛋白III（SGIII），已显示与POMC结合。 SGIII在神经内分泌细胞中发现，并参与将铬烷蛋白A（CGA）运输到RSP中。我们使用RNA干扰（siRNA）在ATT20细胞中击倒SGIII和CPE表达，在两种情况下，我们通过本构分泌途径均通过构成性分泌途径增加了POMC分泌。仅在SGIII敲低细胞中观察到CGA的组成型分泌增加。在双CPE-SGIII敲低细胞中，观察到pOMC的本构分泌增加，并刺激了ACTH的分泌。这些结果表明，CPE参与了POMC贩运到RSP。而且，除了在RSP贩运过程中，SGIII可能在将POMC分类为RSP中为CPE发挥补偿性作用。 我们最近发现，跨膜CPE不仅与小鼠下丘脑中的大型密集核囊泡（LDCV）相关，而且与PC12细胞中小鼠下丘脑中的突触小囊泡（SV）相关。在CPE-KO小鼠中，下丘脑神经元的高K+刺激释放从下丘脑神经元释放。电子显微镜表明，与野生型小鼠相比，CPE-KO小鼠的下丘脑神经元中，突触的活性区域（在活性区的质膜200nm之内）的SV数量显着减少。使用PC12细胞作为模型的总内反射荧光（TIRF）显微镜表明，CPE胞质尾部的过表达降低了含突触素蛋白的突触类细菌的稳态水平，该突触类样的微泡堆积于200 nm以内的区域内，从亚倍膜膜（TIRF ZOIL）（TIRF区域）。我们的发现表明，与γ添加剂和肌动蛋白相互作用的CPE细胞质尾巴是在下丘脑神经元中富含肌动蛋白富的前活性区和PC12细胞TIRF区域中SVS定位的新介体。 我们最近对垂体ATT-20细胞的研究为上调LDCV生物发生的自分泌机制提供了证据，以补充这些囊泡刺激的胞吐作用后的LDCV。自分泌信号被鉴定为Serpinin，这是一种新型的26个氨基酸CGA衍生的肽，它从CGA的C末端裂解。 Serpinin以活性依赖性的方式从LDCV释放。发现分泌的丝丁蛋白激活腺基环化酶以增加cAMP水平，并在细胞中蛋白激酶A。然后，这导致转录因子SP1从细胞质转移到细胞核中，并增加了蛋白酶抑制剂蛋白酶Nexin 1（PN-1）的转录，然后抑制高尔基体复合物中的颗粒蛋白降解。这些蛋白质的稳定在高尔基体中增加了水平，从而显着增强了LDCV的形成。我们还鉴定了垂体ATT-20细胞和心脏组织中的蛇状素，硫酸盐酸盐素（PGLU-serpinin）的N末端修饰形式。发现PGLU-塞铂在ATT-20细胞中具有抗氧化应激的神经保护活性，而大鼠皮质神经元中的K+诱导的凋亡较低。与Bruno Tota博士（卡拉布里亚大学）合作，发现PGLU-Serpinin在心脏功能上具有积极的肌力活性，没有血压和心率的变化。 PGLU-辛替肽通过1-肾上腺素能/腺苷酸环化酶/CAMP/PKA途径起作用。 PGLU-辛替肽和其他CGA衍生的心活性肽作为新颖的 - 肾上腺素能肌力和升性调节剂出现，它们可以一起在心肌如何策划其对交感神教蛋白刺激的复杂反应方面发挥关键作用。 CPE在肥胖症中起着重要作用，最近该基因被创造为肥胖敏感性基因。我们表明，极度肥胖的CPE-KO小鼠的骨矿物质密度低，得出的结论是，缺乏对成熟的货车的处理（一种促进骨形成的肽）可能导致这些小鼠中骨密度不佳的原因。此外，与Lecka-Czernik博士（托莱多大学）合作，我们发现CPE富含来自骨髓的大鼠杂质干细胞系，因此CPE可能参与了另一个水平的调节骨形成。目前正在探索这种可能性。 CPE-KO小鼠的神经系统功能缺乏，包括学习和记忆。我们表明，在6-14周大的CPE-KO小鼠中，树突修剪在皮质和海马神经元中较差，这会影响突触发生。另外，电生理测量显示了这些小鼠海马切片中长期增强（LTP）的缺陷。该缺陷归因于在4周及以上观察到的CPE KO动物海马的CA3区域中神经元的丧失。这些神经元通常在CPE中富含的神经元在三周龄的时候正常。当断奶延迟一周时，直到CPE KO小鼠的产后第5周才观察到这种变性。我们现在已经表明，退化归因于断奶的应力，断奶的压力包括母体分离，耳朵标记和用于基因分型的尾巴剪裁。我们还检查了约束应力对海马神经元中CPE表达的影响。当小鼠接受急性约束应力1H，然后在应激后0、1-24H处死时，它们显示出海马中CPE-MRNA表达的立即和短暂降低。相比之下，轻度慢性应激（7天/天）增加了糖皮质激素的分泌，小鼠在海马中显示出CPE mRNA和蛋白质的增加，并且没有明显的神经元变性。此外，当海马神经元用合成糖皮质激素（地塞米松）处理时，细胞中的CPE mRNA和蛋白质显着增加。这些观察结果表明，CPE的增加可以介导压力期间的神经元存活，并且缺乏CPE会导致神经元变性。 为此，我们在培养大鼠海马神经元中外源性过表达或施用了重组CPE，并显示CPE对这些神经元的存活率和神经保护作用增加，而受到过氧化氢处理或谷氨酰胺细胞毒性的氧化应激。 在小鼠胚胎中检查了CPE的表达，以确定它是否可以在早期胚胎发育中起作用。我们发现，wt cpe和cpe-delta n mRNA早在每天E5.5时表达，并且每天增加，在E8.5达到峰值，在内分泌系统表达之前略微下降到E9.5。 CPE mRNA表达在E 10.5 -11.5至E5.5以下的水平下急剧下降，然后在E12.5时与内分泌系统的发展并行急剧增加，并继续增加到成年。然而，在E10.5时，CPE-DELTAN mRNA最大增加，然后在E11.5-12.5急剧下降，然后少量增加直至PN1。与CPE相反，成年海马不存在CPE-Deltan。原位杂交研究表明，CPE和CPEDELTAN mRNA主要在小鼠胚胎中的前脑和节点中表达。我们已经开始研究使用神经球来研究增殖和分化的神经系统胚胎发育过程中CPE和CPE-delta n的作用。