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: 8351081
• 负责人: Yoke p Loh
• 金额: $ 117.59万
• 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/8351081
• 关键词: Actins; Acute; Adenylate Cyclase; Adverse effects; Affect; Age; Alzheimer' s Disease; Amino Acids; Anabolism; Animals; Anterior Pituitary Gland; Apoptosis; Area; Binding; Biogenesis; Blood Pressure; Bone Density; Bone Marrow; Brain-Derived Neurotrophic Factor; C-terminal; Cardiac; Cell Line; Cell membrane; Cells; Chromogranin A; Chronic stress; Coin; Collaborations; Corticotropin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Tail; Cytoskeleton; Cytosol; Defect; Dense Core Vesicle; Dexamethasone; Diabetes Mellitus; Disease; Dynein ATPase; Electron Microscopy; Endocrine; Endocrine system; Enzymes; F-Actin; Fluorescence; Fluorescence Microscopy; Generations; Genes; Glucocorticoids; Glutamates; Golgi Apparatus; Heart Rate; Heart failure; Hippocampus (Brain); Hormones; Hydrogen Peroxide; Hypothalamic structure; Knockout Mice; Laboratories; Lead; Learning; Long-Term Potentiation; Marketing; Measurement; Mediating; Membrane; Memory; Messenger RNA; Microtubules; Motor; Movement; Mus; Mutation; N-terminal; Nerve Degeneration; Nervous system structure; Neuraxis; Neurodegenerative Disorders; Neuroendocrine Cell; Neurons; Neuropeptides; Obesity; Organelles; Osteogenesis; Oxidative Stress; PC12 Cells; Pathway interactions; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Physiological; Pituitary Gland; Pituitary Hormones; Play; Pro-Opiomelanocortin; Process; Proinsulin; Proprotein Convertase 1; Proprotein Convertase 2; Proteins; Pyroglutamate; RNA Interference; Rattus; Role; Secretory Vesicles; Site; Slice; Small Interfering RNA; Sorting - Cell Movement; Stem cells; Stress; Susceptibility Gene; Synapses; Synaptic Vesicles; Synaptic plasticity; Synaptophysin; System; Tail; Tissues; Transport Vesicles; Universities; Vesicle; Weaning; adducin; autocrine; base; brain cell; carboxypeptidase H; dynactin; heart cell; in vivo; knock-down; knockout animal; mRNA Expression; maternal separation; membrane activity; neuron loss; neuronal survival; neuroprotection; neurotrophic factor; nexin; novel; overexpression; peptide hormone; polypeptide C; prohormone; protein degradation; receptor; secretogranin III; synaptogenesis; trafficking; trans-Golgi Network; transcription factor

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. Transport of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion is critical for endocrine function and synaptic plasticity. We showed that the cytoplasmic tail of a transmembrane form of CPE in hormone or BDNF-containing dense core secretory vesicles plays an important role in their transport to the release site in pituitary cells and hippocampus, respectively. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of endocrine AtT20 cells; and hippocampal neurons; as well as movement of BDNF- and POMC/CPE- vesicles to the processes in these cells. S-tagged CPE tail pulled down microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from AtT20 cell and brain cytosol, indicating their involvement in vesicle transport. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. We also showed that the CPE tail interacted with C-terminus of -adducin, a component of the cytoskeleton that binds and stabilizes F-actin. Overexpression of the C-terminal 38 amino acid of -adducin inhibited the exit of POMC vesicles at the trans-Golgi network. Thus these studies demonstrate that the vesicular CPE cytoplasmic tail plays a novel mechanistic role in facilitating the exit of POMC/ACTH vesicles from the TGN via -adducin/actin interaction; and anchoring POMC and BDNF vesicles to the microtubule-based motor system for transport along the processes to the plasma membrane for activity-dependent secretion in endocrine cells and neurons. We recently found that transmembrane CPE is not only associated with large dense core vesicles, 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 of synapses was significantly decreased in hypothalamic neurons of CPE-KO mice compared with WT mice. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells 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, mediates the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells. We identified a novel 26 amino acid CgA-derived peptide from the C-terminal of CgA that regulates granule biogenesis in an autocrine way. Serpinin, released in an activity-dependent manner from LDCVs, can activate adenyl cyclase to increase cAMP levels, and protein kinase A in the cell leading to expression of a protease nexin 1 (PN1) by the transcription factor sp1Since PN1 inhibits granule protein degradation in the Golgi complex, their stabilization increases 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. Thus pGlu-serpinin is a potential drug for treating heart failure with no side effects seen with other positive inotropic drugs on the market. 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. However, recently, 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 we are currently investigating other possible roles of CPE in bone formation. CPE-KO mice have central nervous system deficiencies, 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 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. Our results demonstrate that the degeneration is correlated with the stress of weaning and maternal separation and that CPE may support neuronal survival. 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), the mice showed an increase in CPE mRNA and protein in the hippocampus, and no neuronal degeneration was evident. This is consistent with a neuroprotective role of CPE. To further investigate this role, we overexpressed CPE in rat hippocampal neurons in culture and found increased survival of these neurons. Additionally when we subjected these CPE overexpressing neurons to oxidative stress with hydrogen peroxide treatment, they were protected against apoptosis. Furthermore, when hippocampal neurons were treated with synthetic glucocorticoid, dexamethasone, there was a significant increase in CPE mRNA and protein in the cells. These findings taken together support our hypothesis that stress-induced secretion of glucocorticoid up-regulates CPE expression in hippocampal neurons to protect them from degeneration in vivo.

我们研究了膜 CPE 和促分泌素 III 作为将 POMC 靶向调节分泌途径 (RSP) 的分选受体的作用。使用我们的 CPE 敲除 (KO) 小鼠，我们发现垂体前叶细胞原代培养物中新合成的 POMC 50% 被降解，这表明由于缺乏 CPE，无法对 RSP 颗粒进行有效分选，POMC 是降解的目标。然而，剩余的一些 POMC 被分类到 RSP 中。补偿性分选受体的候选者是 Secretogranin III (SgIII)，它已被证明可以结合 POMC。 SgIII 存在于神经内分泌细胞中，参与嗜铬粒蛋白 A (CgA) 向 RSP 的运输。我们使用 RNA 干扰 (siRNA) 敲低 AtT20 细胞中 SgIII 和 CPE 的表达，我们证明在这两种情况下，POMC 分泌均通过组成型分泌途径增加。仅在 SgIII 敲低细胞中观察到 CgA 组成型分泌增加。在双 CPE-SgIII 敲低细胞中，观察到 POMC 的组成性分泌增加，并且 ACTH 的刺激分泌受到干扰。这些结果表明 CPE 参与了向 RSP 贩运 POMC； SgIII 除了在 RSP 贩运过程中发挥更一般的作用外，还可能在将 POMC 分类到 RSP 中对 CPE 起到补偿作用。 将激素和 BDNF 囊泡转运至质膜进行活性依赖性分泌对于内分泌功能和突触可塑性至关重要。我们发现，激素或含有 BDNF 的致密核心分泌囊泡中跨膜形式的 CPE 的细胞质尾部在它们分别转运至垂体细胞和海马释放位点的过程中发挥着重要作用。细胞质中CPE胞质尾部的过度表达与内源性尾部竞争减少了内源性POMC、BDNF和荧光标记CPE在内分泌AtT20细胞过程中的定位；和海马神经元；以及 BDNF- 和 POMC/CPE- 囊泡向这些细胞中的过程的移动。 S 标记的 CPE 尾部从 AtT20 细胞和脑细胞质中拉下基于微管的马达、动力蛋白 (p150)、动力蛋白和 KIF1A/KIF3A，表明它们参与囊泡运输。最后，CPE 尾部的过度表达抑制了 AtT20 细胞 ACTH 的调节分泌。我们还表明，CPE 尾部与β-内收蛋白的 C 末端相互作用，β-内收蛋白是结合并稳定 F-肌动蛋白的细胞骨架的一个组成部分。 α内收蛋白 C 端 38 个氨基酸的过度表达抑制了 POMC 囊泡在反高尔基体网络处的退出。因此，这些研究表明，囊泡 CPE 细胞质尾在促进 POMC/ACTH 囊泡通过内收蛋白/肌动蛋白相互作用从 TGN 中退出方面发挥着新的机制作用。将 POMC 和 BDNF 囊泡锚定到基于微管的运动系统，沿着过程运输到质膜，在内分泌细胞和神经元中进行活性依赖性分泌。 我们最近发现跨膜 CPE 不仅与大而致密的核心囊泡有关，还与小鼠下丘脑中的突触小泡（SV）和 PC12 细胞中的突触样微泡有关。在 CPE-KO 小鼠中，高 K+ 刺激的下丘脑神经元谷氨酸释放减少。电镜观察显示，与 WT 小鼠相比，CPE-KO 小鼠下丘脑神经元中位于突触预激活区的 SV 数量显着减少。使用 PC12 细胞的全内反射荧光 (TIRF) 显微镜显示，CPE 胞质尾部的过度表达降低了在距亚质膜 (TIRF 区) 200 nm 范围内积累的含有突触素的突触样微泡的稳态水平。我们的研究结果表明，与γ内收蛋白和肌动蛋白相互作用的CPE胞质尾部介导SV在下丘脑神经元富含肌动蛋白的前活性区和PC12细胞的TIRF区中的定位。 我们从 CgA 的 C 末端鉴定出一种新的 26 个氨基酸的 CgA 衍生肽，它以自分泌方式调节颗粒生物发生。丝氨酸蛋白酶抑制剂以活性依赖性方式从 LDCV 中释放，可以激活腺苷酸环化酶以增加 cAMP 水平，并且细胞中的蛋白激酶 A 导致转录因子 sp1 表达蛋白酶 nexin 1 (PN1)，因为 PN1 抑制细胞中颗粒蛋白的降解。高尔基复合体中，它们的稳定性增加了它们在高尔基体中的水平，从而显着增强了 LDCV 的形成。我们还在垂体 AtT-20 细胞和心脏组织中鉴定了丝氨酸蛋白酶抑制剂的 N 末端修饰形式，即焦谷氨酸丝氨酸蛋白酶抑制剂 (pGlu-serpinin)。 pGlu-丝氨酸蛋白酶抑制剂被发现对 AtT-20 细胞氧化应激和低 K+ 诱导的大鼠皮层神经元细胞凋亡具有神经保护活性。与 Bruno Tota 博士（卡拉布里亚大学）合作，发现 pGlu-serpinin 对心脏功能具有正性肌力活性，而血压和心率没有变化。因此，pGlu-丝氨酸蛋白酶抑制剂是一种治疗心力衰竭的潜在药物，并且没有市场上其他正性肌力药物所见的副作用。 CPE在肥胖中起着重要作用，最近该基因被称为肥胖易感基因。我们发现极度肥胖的 CPE-KO 小鼠骨密度较低，并得出结论认为，缺乏将 pro-CART 加工成成熟 CART（一种促进骨形成的肽）的过程可能是这些小鼠骨密度较差的原因。然而，最近，我们与 Lecka-Czernik 博士（托莱多大学）合作，发现 CPE 在来自骨髓的大鼠间充质干细胞系中富集，因此我们目前正在研究 CPE 在骨形成中的其他可能作用。 CPE-KO 小鼠存在中枢神经系统缺陷，包括学习和记忆。我们发现，在 6-14 周龄的 CPE-KO 小鼠中，皮质和海马神经元的树突修剪很差，这会影响突触发生。此外，电生理学测量显示这些小鼠海马切片的长期增强作用存在缺陷。这种缺陷归因于在 4 周龄及以上观察到的 CPE KO 动物海马 CA3 区域神经元的损失。这些神经元通常富含 CPE，在动物断奶前 3 周龄时表现正常。我们的结果表明，这种退化与断奶和母体分离的压力有关，并且 CPE 可能支持神经元的存活。 我们还研究了限制性应激对海马神经元 CPE 表达的影响。当小鼠受到急性限制性应激1小时，然后在应激后0、1-24小时处死时，它们的海马中CPE-mRNA表达立即且短暂地降低。相比之下，在轻度慢性应激（每天 1 小时，持续 7 天）后，小鼠海马中的 CPE mRNA 和蛋白质增加，并且没有明显的神经元变性。这与 CPE 的神经保护作用一致。为了进一步研究这一作用，我们在培养的大鼠海马神经元中过度表达 CPE，并发现这些神经元的存活率增加。此外，当我们用过氧化氢处理这些 CPE 过度表达的神经元时，它们会受到氧化应激，从而免受细胞凋亡的影响。此外，当用合成糖皮质激素、地塞米松处理海马神经元时，细胞中的 CPE mRNA 和蛋白质显着增加。这些发现共同支持了我们的假设，即应激诱导的糖皮质激素分泌上调海马神经元中的 CPE 表达，以保护它们免受体内退化。