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: 8736792
• 负责人: Yoke p Loh
• 金额: $ 91.24万
• 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/8736792
• 关键词: Adenylate Cyclase; Adrenergic Agents; Adrenergic Receptor; Adult; Affect; Alzheimer' s Disease; Anabolism; Anterior Pituitary Gland; Antiepileptic Agents; Astrocytes; Behavior; Blood Circulation; Blood Pressure; Bone Density; Bone Marrow; Carbamazepine; Cardiac; Cell Culture Techniques; Cell Death; Cell Line; Cell secretion; Cells; Chromogranin A; Chronic stress; Clip; Collaborations; Contracture; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Defect; Development; Dexamethasone; Diabetes Mellitus; Diabetic mouse; Disease; E protein; Ear; Embryo; Embryonic Development; Emotional Stress; Endocrine; Endocrine system; Enzymes; Epilepsy; Exhibits; Fibroblast Growth Factor 2; Genes; Genotype; Gland; Glucocorticoids; Glutamates; Heart Rate; Hippocampus (Brain); Hormones; Hydrogen Peroxide; Hyperglycemia; In Situ Hybridization; Inbred SHR Rats; Inbred WKY Rats; Incubated; Infarction; Ischemia; Knockout Mice; Knowledge; Laboratories; Lead; Learning; Left ventricular structure; Lipids; Mediating; Membrane; Memory; Mental Depression; Mesenchymal Stem Cells; Messenger RNA; Modeling; Mus; Mutation; Myocardium; Names; National Institute of Dental and Craniofacial Research; Neocortex; Nerve Degeneration; Nervous System Physiology; Nervous system structure; Neurodegenerative Disorders; Neuroendocrine Cell; Neurons; Neuropeptides; Obesity; Oligodendroglia; Organelles; Osteoblasts; Osteogenesis; Oxidative Stress; Pathway interactions; Peptides; Pharmaceutical Preparations; Phosphotransferases; Physiological; Pituitary Gland; Pituitary Hormones; Play; Population; Pro-Opiomelanocortin; Process; Proinsulin; Property; Proprotein Convertase 1; Proprotein Convertase 2; Prosencephalon; Proteins; RNA Interference; Rattus; Recombinants; Recovery of Function; Reperfusion Injury; Role; Route; Saliva; Secretory Vesicles; Seizures; Signal Transduction; Site; Somatropin; Somites; Sorting - Cell Movement; Stem cells; Stress; Submandibular gland; Tail; Time; Tissues; Transport Vesicles; Universities; Weaning; adrenergic; carboxypeptidase H; cell type; conditioning; cytotoxicity; depressive symptoms; extracellular; falls; gene therapy; improved; in vivo; knock-down; lipid biosynthesis; mRNA Expression; maternal separation; mutant; nerve stem cell; neurodevelopment; neuron loss; neuronal survival; neuroprotection; neurotrophic factor; normotensive; novel; peptide hormone; prevent; prohormone; receptor; response; restraint stress; secretogranin III; trafficking

We continue to investigate the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway (RSP). CPE knockout (KO) mice show defective trafficking of POMC in primary cultures of anterior pituitary cells since 50% of the newly synthesized POMC was targeted for degradation in the absence of CPE. However, some of the remaining POMC was sorted into the RSP, presumably by Secretogranin III (SgIII). SgIII is found in neuroendocrine cells and is involved in trafficking of chromogranin A (CgA) to the RSP. Our studies using RNA interference to knock down SgIII and CPE showed that both proteins affect the normal secretion behavior of POMC in AtT20 cells, i.e. POMC was secreted at an elevated rate through the constitutive secretory pathway when either CPE or SgIII are reduced. When both are knocked-down, the affect is augmented, suggesting that POMC trafficking is dependent on both proteins for efficient trafficking to the RSP. In other studies of cellular secretion pathways, and in collaboration with Dr. Bruce Baum (NIDCR), we generated a proinsulin mutant (B10-proinsulin) and mutants of human growth hormone (hGH) and expressed them in mouse submandibular glands (SG). These glands provide a delivery route to either the saliva or the circulation. In the case of B10-proinsulin, significant amounts were secreted into the circulation where it functioned to alleviate hyperglycemia in chemically induced diabetic mice. Additionally, we identified a hGH mutant that was secreted into the circulation, in contrast to the WT hGH control, that was secreted almost exclusively into the saliva. This suggests that the SG have the potential to be a target tissue for gene therapy and that the knowledge of sorting determinants of peptide hormones will play a crucial part in this process. 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 beta1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway and emerges as a novel-adrenergic inotropic and lusitropic modulator of the myocardium in response to sympathochromaffin stimulation. We have also investigated the effect of pGlu-serpinin on cardioprotection. Using normotensive (WKY) and hypertensive (SHR) rats as models; we showed that pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection. In both WKY and SHR rats, pGlu-serpinin improved left ventricle function recovery after ischemia. Moreover, it reduced ischemicinduced contracture state and decreased infarct size. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. CPE plays a significant role in obesity and obese CPE-KO mice have low bone mineral density. We showed that the lack of processing of pro-CART to mature CART, a peptide that promotes bone formation, contributes significantly to 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. We found that primary cultures of mouse mesenchymal stem cells that normally differentiate into osteoblasts could be partially switched to a cell type that accumulated lipid when incubated with purified CPE protein. This represents a novel discovery that CPE may contribute to adipogenesis in this pathway. CPE-KO mice have deficiencies in their nervous system function, including learning and memory. This defect is attributed to the loss of neurons in the CA3 region of the hippocampus due to the physical stress of ear tagging and tail clipping for genotyping and the emotional stress of maternal separation at the time of weaning (3 weeks). The neurodegeneration was prevented by the drug carbamazepine, and anti-epileptic drug, demonstrating that the stress associated with weaning induced epileptic-like seizures resulting in neuronal cell death in the absence of CPE. We also examined the effect of restraint stress on CPE expression in hippocampal neurons. When mice were subjected to 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 applied recombinant CPE to 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. Since CPE can act as a neuroprotective molecule with trophic properties, we have recently named it neurotrophic factor-alpha1 (NF-alpha1) to indicate this when CPE functions as a trophic factor. Recently we have investigated the role of CPE in restraint stress-induced depression. Prolonged, but not short-term, stress reduces fibroblast growth factor 2 (FGF2) in the hippocampus, leading to depressive-like behavior in mice. We show in cultured hippocampal neurons that exogenous carboxypeptidase E (CPE/NF-alpha1) up-regulated FGF2 expression independent of its enzymatic activity. In vivo, we found that mice after short-term restraint stress increased hippocampal CPE, FGF2 and doublecortin prior to depressive-like behavior onset. CPE-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the subgranular zone. These mice displayed depressive-like behavior that was rescued by FGF2 administration. Thus, CPE appears to up-regulate hippocampal FGF2 expression during stress, which leads anti-depressant effects. 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-deltaN 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 WT 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 during embryonic development of the nervous system using neurospheres to study proliferation and differentiation. Exogenous addition of recombinant CPE to E13.5 neocortex-derived neurospheres, reduced proliferation of the neurospheres without causing cell death. Furthermore, neurospheres from 7d cultures that were dissociated into single cells and cultured for an additional 5d showed an increase in astrocytes in the presence of CPE, without altering the percentage of neuronal and oligodendrocyte populations. An enzymatically inactive mutant form of CPE was also able to drive differentiation to astrocytes indicating that the enzymatic function of CPE is not essential. Our results suggest a novel role of CPE as an extracellular signal to differentiate neural stem cells into astrocytes.

我们继续研究膜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。 CPE基因敲除（KO）小鼠在垂体垂体细胞的原发性培养物中表现出有缺陷的运输，因为在没有CPE的情况下，有50％的新合成的POMC是降解的目标。但是，剩下的一些POMC被分类为RSP，大概是由章鱼III（SGIII）分类的。 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干扰击倒SGIII和CPE的研究表明，这两种蛋白都会影响pomc在ATT20细胞中的正常分泌行为，即当CPE或SGIII降低CPE或SGIII时，POMC通过本构分泌途径以较高的速率分泌。两者都被击倒后，影响会增加，这表明POMC运输取决于两种蛋白质以有效地运输到RSP。在其他关于细胞分泌途径的研究，以及与Bruce Baum博士（NIDCR）合作的研究中，我们产生了人类生长激素（HGH）的突变蛋白突变体（B10-胰岛素）和突变体，并在小鼠颌下腺（SG）中表达它们。这些腺体提供了通往唾液或循环的送货路线。在B10-胰岛素的情况下，大量分泌到循环中，在该循环中可以减轻化学诱导的糖尿病小鼠的高血糖。此外，我们确定了一个HGH突变体，该突变体被分泌到循环中，与WT HGH控制相比，该突变体几乎完全分泌到唾液中。这表明SG有可能成为基因治疗的靶组织，并且对肽激素的决定因素进行分类的知识将在此过程中起着至关重要的作用。 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BetA1-肾上腺素能受体/腺苷酸环化酶/CAMP/PKA途径起作用，并以副作用刺激刺激响应副热剂的刺激，以心肌的新型 - 肾上腺素能和lusitropic调节剂的形式出现。我们还研究了PGLU-serpinin对心脏保护的影响。使用正常的（WKY）和高血压（SHR）大鼠作为模型；我们表明，PGLU-辛替肽模仿了前调节和调节后诱导的心脏保护。在WKY和SHR大鼠中，PGLU-Serpinin在缺血后改善了左心室功能恢复。此外，它减少了缺血诱导的染色状态并降低了梗塞大小。在PGLU-盐酸素介导的调节后药理学心脏保护中，该机制涉及重新灌注损伤刺激性激酶（风险）途径的激活。 CPE在肥胖症中起着重要作用，肥胖的CPE-KO小鼠的骨矿物质密度低。我们表明，缺乏促销促销手推车的处理，这是一种促进骨形成的肽，对这些小鼠的骨密度差显着贡献。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。我们发现，通常将通常分化成成骨细胞的小鼠间充质干细胞的原发性培养物可以部分切换到与纯化的CPE蛋白一起孵育时积累脂质的细胞类型。这代表了一个新的发现，即CPE可能会导致该途径中的脂肪形成。 CPE-KO小鼠的神经系统功能缺乏，包括学习和记忆。该缺陷归因于海马CA3区域中神经元的丧失，这是由于耳朵标记的身体应力和尾巴剪切的身体应力以及断奶时的母体分离的情绪压力（3周）。神经变性是通过药物甲马西平和抗癫痫药预防的，这表明与断奶诱导的癫痫样癫痫发作相关的压力导致在没有CPE的情况下导致神经元细胞死亡。 我们还检查了约束应力对海马神经元中CPE表达的影响。当小鼠承受轻度的慢性应激（1h/天为7天），这增加了糖皮质激素的分泌时，海马中的小鼠显示出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，我们在培养中将重组CPE应用于大鼠海马神经元，并显示CPE对这些神经元的生存率和神经保护作用增加，当对这些神经元进行氧化应激，并通过过氧化氢处理或谷氨酸盐细胞毒性。由于CPE可以充当具有营养特性的神经保护分子，因此我们最近将其命名为神经营养因子-Alpha1（NF-Alpha1），以表明当CPE充当营养因子时。 最近，我们研究了CPE在约束应力诱导的抑郁症中的作用。延长但不是短期的压力会降低海马中的成纤维细胞生长因子2（FGF2），从而导致小鼠的抑郁样行为。我们在培养的海马神经元中显示了外源羧肽酶E（CPE/NF-Alpha1）上调的FGF2表达与其酶促活性无关。在体内，我们发现短期约束应力后的小鼠在抑郁样行为开始之前会增加海马CPE，FGF2和Doublecortin。 CPE-KO小鼠在亚晶体区域表现出严重降低的海马FGF2水平和未成熟的神经元数。这些小鼠表现出抑郁症状的行为，这些行为是由FGF2给药救出的。因此，CPE在应力过程中似乎上调了海马FGF2表达，从而导致抗抑郁作用。 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-DELTAN mRNA早在每天E5.5时表达，并且每天增加，峰值达到E8.5，在表达内分泌系统之前略微下降到E9.5。 CPE mRNA的表达在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，WT CPE和CPEDELTAN mRNA主要在小鼠胚胎中的前脑和节点中表达。 我们已经开始使用神经球来研究CPE在神经系统的胚胎发育中的作用，以研究增殖和分化。将重组CPE的外源性添加到E13.5新皮层衍生的神经球中，减少了神经球的增殖而不会导致细胞死亡。此外，从7D培养物中分解为单个细胞并培养的5D培养的神经球在存在CPE的情况下显示出星形胶质细胞的增加，而不会改变神经元和少突胶质细胞群体的百分比。 CPE的酶非活性突变体形式也能够驱动与星形胶质细胞的分化，表明CPE的酶促功能不是必不可少的。我们的结果表明，CPE是将神经干细胞区分为星形胶质细胞的细胞外信号的新作用。