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

Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
Niamh X. Cawley, PhD, Staff Scientist
Hong Lou, MD, Senior Research Assistant
Hisatsugu Koshimizu, PhD, Postdoctoral Fellow
Saravana Murthy, PhD, Postdoctoral Fellow
Erwan Thouennon, PhD, Postdoctoral Fellow
Sigrid Young, High School Student
Arlen Papazian, High School Student
Jenny Bhupatkar, MS, Research Assistant
Alicja Woronowicz, MD, PhD, Postdoctoral Fellow
Helen Zhang, Postbaccalaureate Fellow

We study the cell biology of endocrine and neuroendocrine cells. Our focus is three-fold: 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 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 CPE and then secreted. We showed that the sorting of prohormones to the RSP occurs by 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. An RSP sorting receptor that is specific for the sorting signal of these proproteins was identified as the transmembrane form of CPE.

We investigated the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the RSP. Using our CPE knockout (KO) mouse, we showed that about 50% of newly synthesized POMC in primary cultures of pituitary anterior lobe cells was degraded, which suggests that, in the absence of efficient sorting to the granules of the RSP resulting from the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP, as evidenced by its ability to be secreted in a stimulated manner. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII), which has been shown to bind to POMC in precipitation assays. SgIII, is a member of the granin family of proteins, which are found in neuroendocrine cells, and is involved in trafficking of chromogranin A (CgA) to the RSP. We studied its role in the trafficking of POMC to the RSP in AtT20 cells, a pituitary cell line. We used RNA interference (siRNA) to knock down SgIII expression in AtT20 cells and demonstrated that POMC, as well as CgA, was secreted via the constitutive secretory pathway when SgIII was reduced. We also used siRNA to knock down CPE in AtT20 cells and found that reduction of CPE not only raised constitutive secretion of POMC in WT AtT20 cells but added to the elevated constitutive secretion of the POMC found in the SgIII knockdown cells. The constitutive secretion of CgA appeared only marginally affected by the reduction of CPE. 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.

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. Glucagon-like peptide 1 (GLP-1) is an incretin peptide hormone synthesized in intestinal L cells of the gut. It functions to stimulate insulin secretion in a glucose-dependent manner, it slows gastric emptying, and increases β-cell mass. We engineered an adenovirus expressing GLP-1 and transduced murine submandibular salivary gland cells. The transgenic GLP-1 was expressed and secreted into the circulation, where it functioned in an animal model of induced diabetes; mice transduced with the Ad-GLP-1 were protected against the onset of diabetes compared with mice treated with an Ad-Luciferase control virus. Thus, this method of gene therapy for the production of GLP-1 may, in the future, be useful for the treatment of Type 2 diabetes.

Role of CPE in obesity, bone remodeling, neuroprotection, and stress

CPE is required to process pro-neuropeptides to active peptides. We have investigated the role of CPE in the nervous system in vivo. We showed that CPE KO mice were obese and not able to process the precursor of cocaine- and amphetamine-regulated transcript (pro-CART) to CART and therefore lacked this anorexigenic neuropeptide in the hypothalamus. These animals overeat, thus providing further evidence linking a decrease of this neuropeptide to a cause of obesity. We recently showed that CPE KO mice are not only obese, they have low bone mineral density (BMD) accompanied by elevated plasma CTX-1 (carboxy-terminal collagen crosslinks), and osteocalcin, indicators of increased bone turnover. Receptor activator for NF-kappaB ligand (RANKL) expression was elevated approximately two-fold relative to osteoprotegerin in the femur of KO animals, suggesting increased osteoclastic activity in the KO mice. CART, melanocortins, and neuropeptide Y (NPY) in the hypothalamus have been implicated in bone remodeling, given that CART KO mice have low BMD, and MC4R KO and NPY KO mice have increased BMD. However, reduction of alpha-MSH, the primary ligand of MC4R, by up to 94% and the lack of detectable NPY in the hypothalamus of CPE KO do not recapitulate the single-gene KO phenotypes. This study highlights the complex physiological interplay between neuropeptides involved in energy metabolism and bone formation and furthermore suggests the possibility that patients bearing CPE and CART mutations, leading to inactive forms or lack of these molecules, may be at a higher risk for developing osteoporosis. 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 also 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 for 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. 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 suggests 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 hydrogen peroxide–induced oxidative stress. Thus, CPE has a novel neuroprotective role in adult hippocampal neurons. Interestingly, when adult mice were subjected to chronic restraint stress, CPE mRNA and protein were significantly elevated in the CA3 region of the hippocampus, presumably to protect those neurons from degeneration.

CPE mediates peptidergic vesicle transport and synaptic vesicle localization to the pre-active zone for secretion.

Post-Golgi transport of hormone and BDNF vesicles and synaptic vesicle localization to the active zone are mandatory for activity-dependent secretion to mediate endocrine function and synaptic transmission. Using live-cell imaging, we demonstrated 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 is dependent upon the interaction of the vesicular CPE cytoplasmic tail to anchor these organelles to microtubule motors. We have also demonstrated that synaptic vesicles in hypothalamic neurons depend on the interaction of the CPE cytoplasmic tail with g-adducin, which interacts with F-actin to mediate localization of synaptic vesicles to the pre-active zone at the synapse. Our studies show that the cytoplasmic tail of CPE in these vesicles plays a critical role in the organization of hormone and neurotransmitter vesicles to the appropriate position for secretion.

Regulation of secretory granule biogenesis by chromogranin A and serpinin

Formation of large dense-core granules (LDCGs) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our earlier studies on PC12 and AtT-20 cells showed that CgA induces LDCG biogenesis in neuroendocrine cells. CgA is synthesized at the rough ER, then enters the cisternae of this organelle via its signal peptide and is subsequently trafficked to the TGN. There, CgA molecules aggregate at low pH in the presence of calcium; the aggregates provide the physical driving force to induce budding of the TGN membrane, resulting in LDCG formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including our newly identified novel C-terminal peptide, serpinin. Upon stimulation, LDCGs 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 LDCG proteins against degradation in the Golgi complex, thereby enhancing LDCG biogenesis to replenish those that were released. Recently, we showed that the serpinin-driven increase in PN-1 mRNA transcription is mediated via a cAMP-protein kinase A transduction pathway. Both serpinin and 8-Br-cAMP caused translocation of the transcription factor sp1 from the cytoplasm to the nucleus and up-regulation of PN-1 mRNA transcription. This transcription event was blocked by an inhibitor of sp1 binding to specific sites on the PN-1 promoter. Thus CgA and serpinin play important roles in the physical formation and up-regulation of dense-core granule biogenesis, respectively, in (neuro)endocrine cells.

A splice isoform of carboxypeptidase E is a tumor inducer and biomarker for predicting future metastasis.

Metastasis is a major cause of mortality in cancer patients, but the mechanisms governing the complex metastatic process remain elusive. Moreover, there are few prognostic biomarkers for predicting future metastasis/recurrence, and none exists for hepatocellular carcinoma (HCC), a common cancer worldwide, or pheochromocytomas /paragangliomas (PHEO/PGL), rare neuroendocrine tumors. Recently we showed that the obesity susceptibility gene CPE is alternatively spliced to yield a novel gene product (CPE-ΔN) that drives metastasis and is a powerful prognostic biomarker for human cancers. CPE-ΔN mRNA is elevated in cell lines from human metastatic colon, breast, head, and neck and in hepatocellular carcinoma cell lines. SiRNA suppression of CPE-ΔN expression in highly metastatic HCC cells inhibited their growth and metastasis when transplanted into nude mice. In HCC cells, CPE-ΔN is translocated from the cytosol to the nucleus and interacts with histone deacetylase 1/2 to up-regulate expression of various genes, including a metastasis gene, leading to enhanced proliferation and invasion. In retrospective clinical studies of HCC patients, CPE-ΔN mRNA quantification in resected primary tumor (T) and normal surrounding tissue (N) established a cut-off value that predicted recurrence with high sensitivity (90%) and specificity (92%) and was independent of stage. In prospective studies, resected tumor mRNA copy numbers predicted future metastasis/recurrence in PHEO/PGL patients diagnosed with benign tumors at time of surgery with sensitivity and specificity values of 100%. These translational studies demonstrate that CPE-ΔN is a new inducer of tumor metastasis and a powerful prognostic biomarker for predicting future metastasis and recurrence independent of prognosis based on pathohistology in HCC and PHEO/ PGL, cancers of different origins.

Publications

Lou H, Park JJ, Cawley NX, Sarcon A, Sun L, Adams T, Loh YP. Carboxypeptidase E cytoplasmic tail mediates localization of synaptic vesicles to the pre-active zone in hypothalamic pre-synaptic terminals. J Neurochem. 2010 114:886-896.
Cawley NX, Yanik T, Woronowicz A, Chang W, Marini JC, Loh YP. Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density. Am J Physiol Endocrinol Metab. 2010 299:189-197.
Voutetakis A, Cotrim AP, Rowzee A, Zheng C, Rathod T, Yanik T, Loh YP, Baum BJ, Cawley NX. Systemic delivery of bioactive glucagon-like peptide 1 after adenoviral-mediated gene transfer in the murine salivary gland. Endocrinology. 2010 151:4566-4572.
Koshimizu H, Cawley NX, Kim T, Yergey AL, Loh YP. Serpinin: a novel chromogranin A-derived, secreted peptide up-regulates Protease Nexin-1 expression and granule biogenesis in endocrine cells. Mol Endocrinol. 2010 in press.
Murthy SRK, Pacak K, Loh YP. Carboxypeptidase E: elevated expression correlated with tumor growth and metastasis in pheochromocytomas and other cancers. Cell Mol Neurobiol. 2010 in press

Collaborators

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 on 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

Contact

For more information, email lohp@mail.nih.gov or visit scn.nichd.nih.gov.

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