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Secretory Protein Trafficking, Granule Biogenesis, and Cancer in Neuroendocrine Cells

Y. Peng Loh, PhD
  • Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
  • Niamh X. Cawley, PhD, Staff Scientist
  • Hisatsugu Koshimizu, PhD, Postdoctoral Fellow
  • Wensheng Li, PhD, Visiting Scientist
  • Hong Lou, MD, Senior Research Assistant
  • Saravana Murthy, PhD, Postdoctoral Fellow
  • Joshua Park, PhD, Postdoctoral Fellow
  • Trushar Rathod, BS, Postbaccalaureate Fellow
  • Erwan Thouennon, PhD, Postdoctoral Fellow
  • Alicja Woronowicz, MD, PhD, Postdoctoral Fellow
  • Sigrid Young, High School Student
  • Helen Zhang, Postbaccalaureate Fellow

We study the cell biology of endocrine and neuroendocrine cells. Our focus is three-fold: to 1) investigate the mechanisms of biosynthesis and intracellular trafficking of peptide hormones and neuropeptides and their processing enzymes; 2) uncover mechanisms involved in the regulation of dense core secretory granule biogenesis, transport and exocytosis; and 3) determine the physiological and pathological roles of the prohormone processing enzyme carboxypeptidase E (CPE). Our work has led to the discovery of novel molecular mechanisms of protein trafficking to the regulated secretory pathway and identified players and mechanisms that control secretory granule biogenesis and transport in endocrine and neuroendocrine cell as well as uncovered new roles of carboxypeptidase E gene in neuroprotection, dendritic pruning, and cancer. Such studies, using cell lines, primary cell cultures, and mouse models, have provided a better understanding of diseases related to defects in hormone and neuropeptide targeting, synaptic transmission, neurodegeneration, memory, learning, diabetes, obesity, and metastatic disease.

Mechanism of sorting pro-neuropeptides, neurotrophins, and their processing enzymes to the regulated secretory pathway

The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storage, and release of active proteins and peptides in the neuroendocrine cell. We investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin, and brain-derived neurotrophic factor (BDNF) to the RSP. Our studies showed that these pro-proteins undergo homotypic oligomerization, as a concentration step, as they traverse the cell from the site of synthesis in the endoplasmic reticulum (ER) to the trans-Golgi network (TGN), where they are sorted into dense-core granules of the RSP for processing by prohormone convertases and carboxypeptidase E (CPE), and then secreted. We showed that the sorting of prohormones to the RSP occurs via a receptor-mediated mechanism. Site-direct mutagenesis studies have identified a 3-D consensus sorting motif consisting of two acidic residues found in POMC, proinsulin, and BDNF. A RSP sorting receptor that was specific for the sorting signal of these proproteins was identified as the transmembrane form of CPE.

In collaboration with Bruce Baum, we also investigated the secretory behavior of peptide hormones in the exocrine salivary gland. The salivary gland is a target tissue for the expression of proteins for gene therapy, given that it secretes proteins into the upper GI tract via the RSP and into the circulation via the constitutive secretory pathway. Manipulation of sorting signals may be useful in redirecting therapeutic proteins into the circulation. Recently, we demonstrated that mice transduced with an adenovirus construct containing glucagon-like peptide 1 (Ad-GLP-1) in the submandibular gland had serum GLP-1 levels about 3-times higher than did mice treated with the control Ad-Luciferase (Ad-Luc) vector. In fasted animals, glucose levels were similar between Ad-GLP-1 and Ad-Luc treated mice, in keeping with GLP-1’s glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 treated mice cleared the glucose significantly faster than control mice. These studies demonstrate that the bioactive peptide hormone GLP-1, normally secreted from endocrine cells in the gut through the RSP, can be engineered to be secreted into the circulation from exocrine cells of the salivary gland to affect glucose homeostasis.

Role of CPE in obesity, neuroprotection, and stress

Recently, we investigated the role of CPE in the nervous system in vivo. We showed that CPE KO mice were not able to process pro-CART to CART and therefore lacked this anorexigenic neuropeptide in the hypothalamus. These animals overeat, thus providing further evidence linking decrease of this neuropeptide to a cause of obesity. In collaboration with the Accili group, we found that the transcription factor FoxO1 regulates CPE gene expression negatively. Normally insulin binds to insulin receptors in the POMC neurons, which leads to nuclear signaling, nuclear exclusion, and inactivation of FoxO1. To model this physiological event, FoxO1 was deleted in the POMC neurons in the arcuate nucleus of the hypothalamus in mice, which resulted in increased CPE levels, increased α-MSH—an anorexigenic neuropeptide derived from POMC—and reduced food intake, without change in energy expenditure. These findings raise the possibility of targeting CPE to develop weight loss medications.

We demonstrated deficiencies in hippocampal long-term potentiation, learning, and memory in CPE-KO mice. A major cause of this defect was the total degeneration of neurons in the CA3 region of the hippocampus. This was evident only in four-week-old and older CPE-KO mice. Three-week-old KO animals were normal, suggesting that CPE is important in maintaining the survival of CA3 neurons after three weeks of age. Interestingly, we found that the degeneration of the CA3 region that occurs just after three weeks of age could be minimized by delaying weaning of these CPE-KO mice, which normally occurs at three to four weeks of age. However, early weaning at two weeks did not result in degeneration of the CA3 region when observed at three weeks. These results have uncovered a critical period between three and four weeks after birth, when the CA3 neurons are highly sensitive to stress such as maternal separation and weaning, and demonstrated that CPE is required to maintain survival of these neurons after three weeks of age. Indeed, when CPE was overexpressed in hippocampal neurons in culture, the cells were protected from apoptosis after induced oxidative stress using hydrogen peroxide. Thus, CPE has a novel neuroprotective role in adult hippocampal neurons. Other abnormalities in the CPE-KO mouse CNS include enhanced dendrite formation coupled with lack of dendritic pruning. Electron microscopic studies of the hypothalamus of CPE KO mice revealed that 40% of the synapses in the hippocampus lacked pre-synaptic docked synaptic vesicles. In addition, stimulated release of glutamate from embryonic hypothalamic neurons and adult hypothalamic synaptosomes was impaired. These mice also exhibited abnormal glutamate-mediated neurotransmission from the photoreceptors to the inner retina, showing a loss of the b wave in their retinogram. Studies indicate that the CPE tail binds to Rab27A and Rim1, molecules that facilitate synaptic vesicle tethering. Overexpression of CPE tail in neurons reduced synaptic vesicle tethering and hence exocytosis in neurons. Our results show that absence of CPE in the KO mice leads to obesity, neurodegeneration, failure in neurotransmission, deficits in learning and memory, and abnormal behavior.

CPE mediates vesicle transport and exocytosis in endocrine cells and neurons

Post-Golgi transport of hormone and BDNF vesicles for activity-dependent secretion is important in mediating endocrine function and synaptic plasticity. We have demonstrated, using live cell imaging, that the cytoplasmic tail of vesicular transmembrane CPE mediates transport of POMC/ACTH and BDNF vesicles to the secretion site in the endocrine corticotrophic cell line AtT20 and hippocampal neurons, respectively. Pull down experiments using both AtT20-cell and mouse-brain cytosol in vitro showed that the cytoplasmic tail of CPE interacted with the motor adaptor protein dynactin and microtubule motors kinesin-2, kinesin-3, and cytoplasmic dynein. Moreover, competition assays using a CPE tail peptide verified the specificity of the interaction between the CPE tail and dynactin. Thus, the mechanism for the transport of POMC and BDNF vesicles to the release site for activity-dependent secretion in endocrine cells and neurons is dependent upon the interaction of vesicular CPE cytoplasmic tail to anchor these organelles to microtubule motors.

Regulation of secretory granule biogenesis by chromogranin A

Formation of large dense-core granules (LDCGs) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. We showed that chromogranin A (CgA) controls the formation of LDCG in neuroendocrine cells. CgA is synthesized at the rough endoplasmic reticulum (RER) and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and to the TGN, where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane, resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including serpinin, a novel C-terminal peptide. Upon stimulation, DCGs undergo exocytosis, and CgA and its derived peptides are released. Acting extracellularly, serpinin is able to signal the increase in transcription of a protease inhibitor, protease nexin-1(PN-1), which protects DCG proteins against degradation in the Golgi complex, thereby enhancing DCG biogenesis to replenish those that were released. Thus CgA plays a significant role in the formation and regulation of dense core granule biogenesis in (neuro)endocrine cells.

A splice isoform of carboxypeptidase E promotes tumor growth and metastasis

Metastasis is a major cause of mortality in patients with cancer. Identifying molecules that promote tumor metastasis will further elucidate mechanisms underlying the complex metastatic process. We have uncovered a novel splice isoform of CPE (CPE-ΔN) that is elevated in metastatic hepatocellular, colon, breast, prostate, head, and neck carcinoma cells. CPE-ΔN lacks the N-terminus present in secretory granule wild-type CPE and is localized to the nucleus of metastatic cancer cells. Overexpression of CPE-ΔN in hepatocellular carcinoma (HCC) cells promoted their proliferation and migration by up-regulating expression of a metastasis gene via epigenetic mechanisms. SiRNA knockdown of CPE-ΔN expression in highly metastatic HCC cells inhibited their growth and metastasis in nude mice. In retrospective clinical studies, CPE-ΔN RNA quantification in primary HCC and colon tumors from patients established a level that predicted metastasis with high prognostic significance (p<0.0001). Furthermore, in a prospective clinical study of 22 patients with paragangliomas, we were able to predict from the mRNA copy numbers of CPE-ΔN in their resected tumors that three of those patients who were diagnosed as having benign tumors would develop metastasis in the future. Indeed subsequent follow-up of these patients revealed development of metastasis several years later. Thus, CPE-ΔN is a new mediator of tumor metastasis and a powerful prognostic marker.


  • Woronowicz A, Koshimizu H, Chang S-Y, Cawley N, Hill J, Rodriguiz RM, Abebe D, Dorfman C, Senatorov V, Zhou A, Xiong Z-G, Wetsel WC, Loh YP. Absence of carboxypeptidase E leads to adult hippocampal neuronal degeneration and memory deficits. Hippocampus 2008 18:1051-1063.
  • Woronowicz A, Cawley NX, Chang SY, Koshimizu H, Phillips AW, Xiong ZG, Loh YP. Carboxypeptidase E knockout mice exhibit abnormal dendritic arborization and spine morphology in central nervous system neurons. J Neurosci Res 2009 July:[E-pub ahead of print].
  • Park JJ, Koshimizu H, Loh YP. Biogenesis and transport of secretory granules to release site in neuroendocrine cells. J Mol Neurosci 2009 37:151-159.
  • Plum L, Lin H, Dutia R, Jun T, Aizawa K, Matsumoto M, Kim A, Cawley N, Paik J, Loh YP, DePinho R, Wardlaw S, Accili. The obesity susceptibility gene carboxypeptidase E links FoxO1 signaling in hypothalamic pro-opiomelanocortin neurons with regulation of food intake. Nat Med 2009 15:1195-1201.


  • Domenico Accili, MD, Columbia University Medical Center, New York, NY
  • Bruce Baum, DMD, Gene Therapy and Therapeutics Branch, NIDCR, Bethesda, MD
  • Illana Gozez, PhD, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
  • Stephen Hewitt, MD, PhD, Laboratory of Pathology, NCI, Bethesda, MD
  • Terence Lee, PhD, University of Hong Kong, Hong Kong, China
  • Karel Pacak, MD, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
  • Ronnie Poon, MD, PhD, University of Hong Kong, Hong Kong, China
  • Alfred Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD


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