Secretory Protein Trafficking and Granule Biogenesis in Neuroendocrine Cells and Role of Carboxypeptidase E Splice Isoform in Tumor Growth and Metastasis

Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
Niamh X. Cawley, PhD, Staff Scientist
Hisatsugu Koshimizu, PhD, Postdoctoral Fellow
Saravana Murthy, PhD, Postdoctoral Fellow
Joshua Park, PhD, Postdoctoral Fellow
Andre Phillips, PhD, Postdoctoral Fellow
Alicja Woronowicz, MD, PhD, Postdoctoral Fellow
Irina Arnaoutova, PhD, Guest Researcher
Hong Lou, MD, Senior Research Assistant
Trushar Rathod, BS, Postbaccalaureate Fellow
Helen Zhang, Degree, Postbaccalaureate Fellow
Nipun Sodhi, High School Student
Sigrid Young, High School Student

We study the cell biology of endocrine and neuroendocrine cells, focusing on the mechanisms of biosynthesis and intracellular trafficking of peptide hormones and neuropeptides and their processing enzymes, the regulation of dense-core secretory granule biogenesis, and the physiological roles of the prohormone processing enzyme carboxypeptidase E. We discovered 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 cells and neurons. Using cell lines, primary cell cultures, and mouse models, we have gained a better understanding of diseases related to defects in hormone and neuropeptide targeting, synaptic transmission, memory, and learning and of diabetes and obesity. Recently, we discovered a splice isoform of carboxypeptidase E (CPE-ΔN) that is highly expressed in metastatic tumor cells. We are investigating the mechanism by which CPE-∆N promotes tumor growth and metastasis as well as the use of CPE-∆N as a biomarker for metastatic potential of primary tumors and a therapeutic target for malignant tumors. Studies in human tumor cell lines, primary and metastatic tumors, and mouse models have provided new insights into the mechanism of tumor metastasis, facilitating the identification of a potential therapeutic target for suppressing tumor metastasis.

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

Cawley, Lou, Rathod, Young, Loh; in collaboration with Baum

The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for the 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. We showed that, in a concentration step, these pro-proteins undergo homotypic oligomerization as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN), where they are sorted into dense-core granules of the RSP for processing by prohormone convertases (PC) and carboxypeptidase E (CPE) and then secreted. Site-directed mutagenesis studies identified a consensus sorting motif consisting of two acidic residues 12 to 15Å apart exposed on the surface of the pro-proteins molecules and two hydrophobic residues at a distance of 5 to 7Å from the acidic residues, with both residue types needed for sorting to the RSP. BDNF, which is secreted in an activity-dependent manner, has such a consensus sorting motif, but constitutively secreted nerve growth factor (NGF) lacks one amino acid residue for completion of the motif. Introduction of the missing residue by mutagenesis (Val20Glu) redirected NGF to the RSP, further confirming the importance of the sorting motif in targeting to the RSP.

We identified an RSP sorting receptor specific for the sorting signal of POMC, proinsulin, and BDNF as the transmembrane form of CPE. The two acidic residues in the prohormone/pro–BDNF sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor CPE to bring about sorting to the RSP. Using a CPE knockout (KO) mouse model, we showed that BDNF was not sorted to the RSP but rather was secreted constitutively in cortical and hippocampal neurons of the CPE KO mice. We observed that, in these mice, constitutive secretion of proinsulin from isolated pancreatic islets was elevated and plasma levels of proinsulin were significantly higher, indicating a role of CPE in the intracellular trafficking of proinsulin. Recent studies of POMC sorting in primary anterior pituitary cultures from CPE KO mice showed a defect in the regulated secretion of ACTH, consistent with CPE’s role as a sorting receptor in vivo; however, we did observe some stimulated secretion of POMC.

Further investigation of the pituitaries revealed elevated expression of secretogranin III (SgIII), a lipid raft–associated molecule found in the TGN. Previous work reported in the literature has shown that SgIII binds to POMC, albeit with lower affinity than to CPE, and can potentially act as a sorting receptor for POMC. Thus, SgIII appears to be expressed at higher levels as a compensatory sorting receptor for POMC in some CPE KO mice. Our studies provide evidence of a sorting signal/receptor–mediated mechanism for targeting prohormones, pro-neuropeptides, and the neurotrophin BDNF to the RSP in endocrine cells and neurons.

In collaboration with Bruce Baum, we 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 as 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 directing therapeutic proteins into a specific pathway. Previously, we generated a bioactive mutant form of human growth hormone (hGH) and demonstrated that we could partially redirect it into the constitutive pathway and thus to the circulation of rats. More recently, the fusing of hGH to erythropoietin (Epo), a protein secreted constitutively from salivary gland cells, allowed the Epo to be targeted to the saliva via the RSP in both mouse and rat model systems, demonstrating a proof of concept that a therapeutic protein without an RSP sorting signal can be directed into the RSP by coupling it with hGH. We took a similar approach with proinsulin and found that a mutant proinsulin, which does not sort efficiently to the RSP in endocrine cells but is secreted constitutively, is secreted into the circulation and produces glucose-lowering effects in mice.

Cawley NX, Arnaoutova I, Yanik T, Lou H, Patel N, Loh YP. Techniques in neuropeptide processing, trafficking, and secretion. In: Gozes I, ed. Methods in Neurobiology. Humana Press, 2007;67-96.
Samuni Y, Cawley NX, Zheng C, Cotrim AP, Loh YP, Baum BJ. Sorting behavior of a transgenic erythropoietin–growth hormone fusion protein in murine salivary glands. Hum Gene Ther 2008;19:279-286.
Samuni Y, Zheng C, Cawley NX, Cotrim AP, Loh YP, Baum BJ. Sorting of growth hormone-erythropoietin fusion proteins in rat salivary glands. Biochem Biophys Res Commun 2008;373:136-139.

The CPE knockout mouse exhibits neurodegeneration, behavioral deficits, and aberrant synaptic transmission

Woronowicz, Koshimizu, Cawley, Lou, Murthy, Loh; in collaboration with Wetsel

By deleting exons 4 and 5 from the CPE gene, we generated CPE KO mice and characterized their phenotype. The KO mice became obese by 10 to 12 weeks of age and weighed 60 to 80 g by 40 weeks. The null animals consumed more food, were less physically active during the light phase of the light-dark cycle, and burned fewer calories as fat than wild-type (WT) littermates. The animals’ fasting levels of glucose and insulin-like immunoreactivity (IR) in plasma were elevated at around 20 weeks; males recovered from the elevated levels by 32 weeks while the females did not. At 32 weeks, the plasma insulin-like IR, which consists of primarily proinsulin, was 50 to 100 times higher in the KO mice than in WT animals. The KO mice showed impaired glucose clearance and were insulin-resistant. They also exhibited high levels of leptin in plasma but showed no circulating fully processed CART, a peptide responsive to leptin-induced feedback inhibition of feeding. Besides the obesity and diabetes phenotypes, the KO mice were subfertile and showed deficits in GnRH processing in the hypothalamus.

Behavioral analyses revealed that adult KO mice had diminished reactivity to stimuli and reduced toe-pinch reflexes, muscle strength, coordination, and visual placing. Moreover, they showed delayed learning and poor memory consolidation in the water-maze and object-preference tests. Electrophysiological studies indicated an inability to generate long-term potentiation when the CA1 region of the hippocampus was stimulated. Analysis of brain sections revealed that, at 4 weeks of age and older, the KO mice had an abnormal hippocampal structure, including the absence of the CA3 region. Interestingly, postnatal KO animals at 3 weeks and younger showed a normal hippocampus. Thus, 3 weeks is a critical age at which the CA3 region undergoes degeneration in the absence of CPE. Moreover, the mossy fibers terminated prematurely before reaching the CA1 region. Interestingly, Sholl analysis of hippocampal CA1 region neurons showed that the complexity of dendritic arborizations of neuronal cells is more pronounced in CPE-KO mice than in WT littermates, suggesting enhanced dendrite formation coupled with lack of dendritic pruning. Our observations indicated that, in the adult animal, CPE plays an essential role in maintaining neuronal survival and the structure of the hippocampus. We further demonstrated the neuroprotective role of CPE in ex vivo experiments. Overexpression of CPE in hippocampal neurons resulted in protection of the neurons from apoptosis after hydrogen peroxide–induced oxidative stress.

Electron microscopic studies of the hypothalamus revealed additional morphological abnormalities in the CPE-KO mouse. Forty percent of the synapses in the hippocampus of the CPE KO mice showed an absence of presynaptic docked synaptic vesicles. In addition stimulated release of glutamate from embryonic hypothalamic neurons and adult hypothalamic synaptosomes was impaired. The mice also exhibited abnormal glutamate-mediated neurotransmission from the photoreceptors to the inner retina, showing a loss of the b wave in their retinogram. Thus, CPE appears to play a critical role in synaptic transmission, possibly by facilitating synaptic vesicle docking and exocytosis at the synapse (see preliminary evidence below). Absence of CPE in the KO mice therefore leads to failure in neurotransmission, deficits in learning and memory, and abnormal behavior and neurodegeneration.

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.

Role of CPE in vesicle transport, docking, and secretion in endocrine cells and neurons

Park, Lou, Phillips, Cawley, Loh

Post-Golgi transport of hormone and BDNF vesicles for activity-dependent secretion is important in mediating endocrine function and synaptic plasticity. We examined the role of the cytoplasmic tail of vesicular transmembrane CPE in the transport of POMC/ACTH and BDNF vesicles in the endocrine corticotrophic cell line AtT-20 and hippocampal neurons, respectively. Overexpression of the CPE cytoplasmic tail diminished localization of endogenous POMC and BDNF in the processes of AtT-20 cells and hippocampal neurons. Furthermore, live-cell imaging showed that overexpression of the CPE tail decreased both the velocity and processivity of POMC and BDNF vesicle movement in live primary anterior pituitary cells and hippocampal neurons. Our findings demonstrated that the CPE tail is involved in the processive trafficking of POMC and BDNF vesicles to the plasma membrane for secretion. We then performed pulldown experiments using both AtT-20 cell and mouse brain cytosol in vitro and 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 specific 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 requires the interaction of vesicular CPE cytoplasmic tail to anchor these organelles to the microtubule motors.

Recently, we demonstrated in CPE KO mice that high K⁺–stimulated secretion of glutamate was absent from cultured primary embryonic hypothalamic neurons (E16) and adult mouse synaptosomes, suggesting the presence of CPE in WT glutamate vesicles and CPE’s involvement in mediating the release of glutamate. Indeed, subcellular fractionation studies revealed that a synaptophysin-enriched synaptic vesicle fraction from mouse brain contained both CPE and the vesicular glutamate transporter Vglut2, but not the dense-core granule–specific protein marker chromogranin A (CgA). Moreover, intact synaptic vesicles could be labeled with extreme C-terminal CPE IgGs but not with non-specific IgGs. As in the case of peptidergic dense-core vesicles, our findings pointed to the existence of a population of synaptic vesicles that contain transmembrane CPE with a cytoplasmic tail. In GST (glutathione-S-transferase) pulldown assays of mouse brain cytosol, we found that GST tagged the CPE cytoplasmic tail CPE_C10 and bound to Rab27A and Rim1, proteins necessary for vesicle docking to the plasma membrane. Thus, our findings suggest a function of the vesicular CPE cytoplasmic tail in the recruitment of Rab27A and Rim1 for docking of glutaminergic vesicles to the plasma membrane for exocytosis in the hypothalamus.

Yeast two-hybrid studies uncovered additional functions of the CPE cytoplasmic tail and showed that the CPE tail interacted with γ-adducin, as confirmed in precipitation studies in vitro. It is known that γ-adducin is primarily a component of the cytoskeleton that binds to and stabilizes F-actin and that it mediates actin binding to fodrin. To assess the significance of the CPE tail–γ-adducin interaction in a functional system, we overexpressed the last 25 amino acids of the CPE C-terminal domain (CPE25) in the cytoplasm of AtT-20 cells. Overexpression of the CPE_C25 peptide reduced localization of γ-adducin to the processes of AtT-20 cells, suggesting that the interaction with the CPE cytoplasmic tail targets γ-adducin to the cells’ processes. It is possible that γ-adducin binds to one of the vacant CPE cytoplasmic tails on the vesicles that are transported to the processes by microtubule-based motors. We hypothesize that, linked to the CPE tail, γ-adducin participates in the tethering of ACTH vesicles to cortical actin just below the plasma membrane in readiness for priming, docking, and release upon stimulation.

Park JJ, Cawley NX, Loh YP. A bi-directional carboxypeptidase E-driven transport mechanism controls BDNF vesicle homeostasis in hippocampal neurons. Mol Cell Neurosci 2008;39:63-73.
Park JJ, Cawley NX, Loh YP. Carboxypeptidase E cytoplasmic tail-driven vesicle transport is key for activity-dependent secretion of peptide hormones. Mol Endocrinol 2008;22:989-1005.
Park JJ, Loh YP. How peptide hormone vesicles are transported to the secretion site for exocytosis. Mol Endocrinol 2008;July 31;22:151-159.

Regulation of secretory granule biogenesis by a chromogranin A–derived peptide

Koshimizu, Cawley, Loh; in collaboration with Yergey

Formation of large dense-core granules (LDCG) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our previous studies uncovered an on/off switch, chromogranin A (CgA), that controls the formation of LDCGs in neuroendocrine cells. Depletion of CgA in rat PC12 cells using antisense technology resulted in the loss of both LDCGs and regulated secretion and in degradation of granule proteins, including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the WT phenotype. In 6T3, a mutant endocrine cell line lacking CgA, LDCGs, and regulated hormone secretion, transfection of CgA resulted in the rescue of granule biogenesis and the RSP.

The importance of CgA in LDCG biogenesis was evident not only in cell lines but also in vivo. In the adrenal medulla of an antisense mRNA transgenic mouse model deficient in CgA, we observed both quantitative and qualitative severely aberrant granule formation as well as a correlation between the amount of depletion of CgA and the reduction in secretory granule biogenesis. Reduction in granule proteins accompanied the reduction in secretory granule biogenesis in both the adrenal medulla of the CgA-deficient transgenic animals and 6T3 cells lacking CgA. Using 6T3 cells lacking secretory granules as a model, we showed that the reduced granule protein levels were attributable to degradation occurring at the Golgi apparatus.

We therefore proposed that regulation of the stability of granule proteins at the Golgi apparatus by CgA is a focal point for control of granule biogenesis in neuroendocrine cells. In support of our hypothesis, we have found in the Golgi, protease nexin-1 (PN-1), a protease inhibitor that is transcriptionally activated by CgA and upregulated in cells actively forming LDCGs; however, PN-1 is downregulated in cells minimally expressing CgA and exhibiting low levels of LDCG biogenesis. Moreover, transfection of PN-1 into 6T3 cells lacking CgA prevented LDCG protein degradation and rescued granule biogenesis. Furthermore, downregulation of expression of PN-1 by antisense RNA in 6T3 cells transfected with CgA resulted in enhanced degradation of granule proteins and reduced secretory granule formation. Recently, we showed that 6T3 cells incubated with conditioned medium from 6T3-bCgA cells or AtT-20 cells exhibited an increase in PN-1 mRNA and granule biogenesis. Likewise, stimulation of AtT-20 cells with high potassium also resulted in an increase in PN-1 mRNA, which was blocked by actinomycin D. We hypothesized that secreted CgA, or a fragment of CgA, might signal the cell to increase levels of PN-1 in response to reduced LDCGs after stimulated secretion. We have now identified a 26–amino acid C-terminal fragment of CgA in the secretion medium of AtT-20 cells, which we named serpinin, that enhanced PN-1 transcription and granule biogenesis in 6T3 cells. Serpinin was elevated in the medium after high K+ stimulation of AtT-20 cells. Thus, we have discovered that serpinin, a new CgA-derived peptide, is co-secreted with POMC-derived hormones upon stimulation of pituitary corticotrophs and signals replenishment of LDCGs by transcriptionally upregulating the expression of PN-1. PN-1 then stabilizes granule proteins, leading to increased LDCG biogenesis.

Kim T, Gondre-Lewis M, Arnaoutova I, Cawley NX, Loh YP. Neurosecretory protein trafficking and dense-core granule biogenesis in neuroendocrine cells. In: Lajtha A, Banik N, eds. Handbook of Neurochemistry and Molecular Neurobiology, 3rd Edition. Neural Proteins: Metabolism and Function. Springer Verlag 2007;10:153-168.
Park JJ, Koshimizu H, Loh YP. Biogenesis and transport of secretory granules to release site in neuroendocrine cells. J Mol Neurosci 2008; [E-pub ahead of print].

Role of Aquaporin 1 in hormone secretion and secretory granule biogenesis

Arnaoutova, Cawley, Rathod, Kim, Loh

We found that the water channel protein aquaporin 1 (AQP1), which is normally present in the plasma membrane, is also localized in secretory granules of endocrine cells. Moreover, when we transfected 6T3 cells lacking CgA with CgA, which rescues granule biogenesis in these cells, the expression of AQP1 mRNA and protein rose significantly. To investigate the role of AQP1 in hormone sequestration and granule biogenesis, we stably transfected AtT-20 cells with an antisense construct of AQP1 to downregulate expression of the protein. AQP1-deficient AtT-20 cells showed a dramatic reduction of secretory granules. Pulse-chase studies demonstrated a defect in regulated secretion of the endogenous ACTH hormone as well as increased degradation of newly synthesized granule proteins such as POMC and CPE at the Golgi apparatus. However, the deficiency of AQP1 in these cells did not affect the transcription and translation of the proteins. AQP1 appears to be critical for the mechanism of secretory granule exocytosis. A defect in secretion leads to a feedback to down-regulate secretory granule biogenesis. We used AQP1-knockout mice in vivo in cellular studies to verify our conclusions. Examination of the animals’ pituitary content and serum revealed lower levels of ACTH in tissue and the circulation than in control mice, consistent with deficits in secretion and storage of ACTH in granules of the pituitary gland. Thus, AQP1 is necessary for maintaining hormone secretion and granule biogenesis in endocrine cells.

Arnaoutova I, Cawley NX, Patel N, Kim T, Rathod T, Loh YP. Aquaporin 1 is important for maintaining secretory granule biogenesis in endocrine cells. Mol Endocrinol 2008;22:1924-1934.

Carboxypeptidase E regulates growth and metastasis in tumor cell lines

Murthy, Cawley, Lou, Loh; in collaboration with Lee, Poon, Ng, Hewitt, Pacak

Elucidation of molecules that control cancer cell growth and invasion will greatly facilitate the identification of biomarkers that can predict impending tumor metastasis while providing targets for therapy. Despite the huge repertoire of biomarkers reported to be useful in identifying aggressive tumors and predicting prognosis, for most cancers there is a dearth of reliable markers for predicting future metastasis from biopsies or resected primary tumors. Recently, we identified a new molecule, an alternatively spliced isoform of carboxypeptidase E (CPE-ΔN) that lacks the N-terminus present in the WT CPE. Human tumor cell lines from liver, colon, breast, prostate, colon, head, and neck that are highly metastatic showed higher expression of CPE-ΔN than did matched tumor lines with low metastatic potential. We also demonstrated that CPE-ΔN plays a role in promoting growth and cell invasion in human cancer cells. Overexpression of CPE-ΔN in the low-metastatic human hepatocellular carcinoma cells (HCC) MHCC97L significantly increased cell proliferation and migration by upregulating expression of a metastasis gene. When si-RNA downregulated CPE-ΔN expression by 56 to 85 percent in highly metastatic cell lines from breast (MDA-MB-23), prostate (DU145), head and neck (MDA 1986), colon (HT29), and liver (MHCC97M3), the si–CPE treated cells showed 56 to 85 percent inhibition. We used the matrigel invasion assay to assess the invasion capability of the tumor cell lines transduced with CPE si-RNA. In all the tumor lines, suppression of CPE expression led to a 70 to 85 percent inhibition of invasion. To complement these observations, we performed in vivo animal studies. In one study, we injected nude mice subcutaneously with MHCCLM3 cells transduced with either si-CPE-ΔN or si-scr. Thirty days after cell inoculation, control mice bearing the si-scr MHCCLM3 cells had much larger liver tumors than did mice injected with si-CPE-ΔN cells. Using a metastatic orthotopic nude mouse model, we subcutaneously injected into the animals’ flank MHCCLM3 cells transduced with either si-scr or si-CPE-ΔN. We then removed the subcutaneous tumor, cut it into 1 to 2 mm cubes, and implanted it into the liver of nude mice. Thirty-five days post-implantation, mice with the si-scr-MHCCLM3–derived tumors developed intrahepatic metastasis and extrahepatic metastasis to the lung while mice inoculated with si-CPE-ΔN-MHCCLM3–derived tumors had smaller tumors and failed to demonstrate metastasis. Our in vitro and in vivo results demonstrated that CPE-ΔN is a newly discovered mediator of metastasis of several human tumor cells.

Clinical use of CPE-∆N as a prognostic biomarker for metastasis and a therapeutic target

Murthy, Loh; in collaboration with Hewitt, Pacak, Lee, Poon, Ng

To determine if CPE-ΔN is a useful biomarker for predicting recurrence and metastasis, we analyzed two retrospective cohorts of patients in blinded studies. Using quantitative RT-PCR and Western blots, we measured CPE-ΔN mRNA and protein levels in primary tumors from HCC and colon cancer patients. RT-PCR verified that only CPE-ΔN mRNA but not WT CPE mRNA was expressed in primary HCC or colonic tumors. Using qRT-PCR, we compared CPE-ΔN mRNA in the primary tumor (T) with that in surrounding non-tumor (N) tissue and determined the ratio (T/N). In 89.8 percent of HCC patients who were disease-free one year after surgery, their CPE-ΔN mRNA ratio was less than or equal to 2, whereas 92 percent of patients with extra- or intrahepatic metastasis/recurrence exhibited a T/N ratio greater than 2. The Kaplan-Meier plot of disease-free survival of 99 HCC patients showed shorter survival times when CPE-ΔN mRNA T/N was greater than 2 in the primary tumor compared with patients with T/N less than or equal to 2. We obtained similar results for 68 colon cancer patients. Western blots also showed significantly higher CPE-∆N levels in HCC patients with recurrence than in the disease-free group. Immunohistochemistry of CPE-ΔN in HCC tumors from 37 HCC patients revealed immunostaining primarily in the nuclei of tumor cells in patients who subsequently developed recurrence; the immunostaining was absent in cell nuclei of patients who remained disease-free. We found the intensity of immunostaining, as determined by image analysis, to differ statistically between the groups. Thus, CPE-ΔN is a powerful prognostic biomarker for predicting future metastasis not only for HCC or colonic but potentially for other types of cancer as well.

Collaborators

Bruce Baum, DMD, Gene Therapy and Therapeutics Branch, NIDCR, Bethesda, MD
Stephen Hewitt, MD, PhD, Laboratory of Pathology, NCI, Bethesda, MD
Joanna Hill, PhD, Laboratory of Behavioral Neuroscience, NIMH, Bethesda, MD
Terence Lee, PhD, University of Hong Kong, Hong Kong, China
Irene Ng, MD, 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
William Wetsel, PhD, Duke University, Durham, NC
Alfred Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

For further information, contact lohp@mail.nih.gov or visit http://scn.nichd.nih.gov.