Valérie Turquier

Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells ROLE OF CONSERVED N- AND C-TERMINAL PEPTIDES

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.

PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells

To determine the possible role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in the development of the sympathoadrenal cell lineage, we have examined the effects of this neurotrophic peptide, in comparison to nerve growth factor (NGF), on the morphology, electrophysiological properties, expression of neuronal and neuroendocrine marker genes, and activity of transcription factors during differentiation of sympathoadrenal‐derived cells, using the rat pheochromocytoma PC12 cell model. Both PACAP and NGF elicited rapid neurite outgrowth, which was accompanied by induction of cell excitability and the development of both sodium and calcium currents. Concurrently, PACAP and NGF increased the expression of a marker of synaptic vesicles. By contrast, PACAP, but not NGF, regulated the expression of different constituents of neuroendocrine large dense core vesicles in PC12 cells. Furthermore, PACAP and NGF differentially regulated the expression of mammalian achaete‐scute homologue and paired homeobox 2b genes, transcription factors instrumental for sympathoadrenal development. To compare downstream effectors activated by PACAP and NGF, we studied the effects of these factors on the binding activity of consensus 12‐O‐tetradecanoylphorbol‐13‐acetate‐ and cAMP‐responsive elements to nuclear extracts of differentiating PC12 cells. We found that both PACAP and NGF markedly increase the binding activity of these cis‐regulatory sequences and that PACAP preferentially recruits activator protein‐1‐like transcription factors to these elements. Taken together, these results show that PACAP and NGF exert common as well as different effects on neuronal and neuroendocrine traits in differentiating PC12 cells, strongly suggesting that these two trophic factors could play complementary roles in the development of the sympathoadrenal cell lineage.

Role of PACAP in the physiology and pathology of the sympathoadrenal system

Sympathetic neurons and chromaffin cells derive from common sympathoadrenal precursors which arise from the neural crest. Cells from this lineage migrate to their final destination and differentiate by acquiring a catecholaminergic phenotype in response to different environmental factors. It has been shown that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its PAC1 receptor are expressed at early stages of sympathetic development, and participate to the control of neuroblast proliferation and differentiation. PACAP also acts as a neurotransmitter to stimulate catecholamine and neuropeptide biosynthesis and release from sympathetic neurons and chromaffin cells, during development and in adulthood. In addition, PACAP and its receptors have been described in neuroblastoma and pheochromocytoma, and the neuropeptide regulates the differentiation and activity of sympathoadrenal-derived tumoral cell lines, suggestive of an important role in the pathophysiology of the sympathoadrenal lineage. Transcriptome studies uncovered genes and pathways of known and unknown roles that underlie the effects of PACAP in the sympathoadrenal system.

Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II‐derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66‐immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9–3.7) ng/ml and 2.9 (1.9–4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10‐fold higher than those of healthy volunteers (26.9 (7.3–44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2–3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma.

Molecular characterization of frog chromogranin B reveals conservation of selective sequences encoding potential novel regulatory peptides1

Chromogranin B (CgB) is a member of the granin family of neuroendocrine secretory proteins, which has been proposed to play a role in secretory granule biogenesis and as a precursor to bioactive peptides. The cloning of CgB in a phylogenetically distant vertebrate, the frog Rana ridibunda, reveals a modest overall homology (35–40%) with mammalian CgB. However, the sequences of the N‐ and C‐terminal regions are more highly conserved (57–65% amino acid identity) and may give rise to novel regulatory peptides. In frog, intense expression of CgB mRNA was observed in particular structures of the brain and in the distal lobe of the pituitary.

Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells.

Secretoneurin (SN) and EM66 are two highly conserved peptides that derive from the processing of secretogranin II (SgII), one of the major constituents of chromaffin cell secretory vesicles. It has been shown that PACAP regulates SgII gene transcription and SN release in bovine adrenochromaffin cells. The aim of the present study was to localize and characterize EM66 in the bovine adrenal gland, and to examine the signaling pathways activated by PACAP to regulate the secretion of EM66 from cultured chromaffin cells. Double immunohistochemical labeling showed an intense EM66-immunoreactive (EM66-IR) signal in TH-positive medullary chromaffin cells of the adrenal gland. HPLC analysis combined with RIA detection revealed, in adrenal medulla extracts and cultured chromaffin cell media, the presence of a major EM66-IR peak co-eluting with the recombinant peptide. PACAP dose-dependently stimulated EM66 release from chromaffin cells (ED(50)=4.8 nM). The effect of PACAP on EM66 secretion was observed after 6 h of treatment and increased to reach a 2.6-fold stimulation at 48 h. The nonselective calcium channel blocker NiCl(2), the cytosolic calcium chelator BAPTA-AM and the L-type calcium channel blocker nimodipine significantly inhibited the stimulatory effect of PACAP on EM66 release. The secretory response to PACAP was also significantly lowered by the protein kinase A inhibitor H89 and by the protein kinase C inhibitor chelerythrine. Concomitant administration of chelerythrine, H89, NiCl(2) and BAPTA totally abolished PACAP-stimulated EM66 secretion. The MAPK inhibitors U0126 and SB203580 respectively decreased by 63% and 72% PACAP-evoked EM66 release. These results indicate that, in bovine adrenal medulla, SgII is processed to generate the EM66 peptide and that PACAP activates multiple signaling pathways to regulate EM66 release from chromaffin cells, suggesting that EM66 may act downstream of the trans-synaptic stimulation of the adrenal medulla by neurocrine factors.

Characterization of Chromogranins in the Frog Rana ridibunda

Up to now, investigations on the function of chromogranins have been conducted mainly in mammalian species. Although much has been learned from these studies on the structure, properties and distribution of chromogranins, their physiological function is still a matter of debate. Studies in distant vertebrate species will bring novel information that may help to solve the enigma of chromogranin function. From the cloning of the frog SgII and CGA proteins, we could learn that evolutionary pressure has acted to conserve the sequence of specific amino acid stretches within these proteins. The fact that some of these conserved regions have been characterized as free peptides such as SN or vasostatin for which biological activities have been described, strongly suggests that one of the functions of chromogranins is to serve as precursors to biologically active peptides. This notion still needs further support, for example by characterization of the receptors for these peptides. On the other hand, chromogranins exhibit a high content of acidic amino acids in all species studied to date, suggesting that this property is probably important for their function. It is conceivable that before chromogranins are released from the cells to act as neuropeptides or hormones, they may participate in the intracellular events leading to secretory granule formation as has been previously suggested (Huttner and Natori 1995). Their highly acidic nature could be the basis for such a complementary role.

PACAP Stimulates the Release of the Secretogranin II‐Derived Peptide EM66 from Chromaffin Cells

Abstract:  The aim of the present article was to examine the effect of PACAP on the release of the SgII‐derived peptide EM66 from primary cultures of bovine chromaffin cells. PACAP dose dependently stimulated EM66 release from cultured chromaffin cells. A significant response was observed after 6 h of treatment with PACAP and increased to reach a 3.6‐fold stimulation at 72 h. The stimulatory effect of PACAP was mediated through multiple signaling pathways, including calcium influx through L‐type channels, PKA, PKC, and MAP‐kinase cascades, to regulate EM66 release from chromaffin cells. These data suggest that EM66 may act downstream of the trans‐synaptic stimulation of the adrenal medulla by neurocrine factors.

Chromogranins/Secretogranins and Derived Peptides: Insights from the Amphibian Model

ABSTRACT Chromogranins/secretogranins (Cg/Sg) are a class of acidic, secretory proteins that occur in endocrine, neuroendocrine, and neuronal cells. Cloning and characterization of chromogranin A, chromogranin B, and secretogranin II revealed that selective sequences within these proteins, bounded by potential processing sites, have been remarkably conserved between amphibian and mammalian species. Two peptides, named secretoneurin and vasostatin, which occur in these regions, have been shown to exert various biological activities. Cg/Sg may also be involved in the formation of secretory granules in amphibians as in mammals. Recent results obtained on the function of Cg/Sg as propeptide precursors and as granulogenic factors in different vertebrate species are discussed.

Proinflammatory Cytokines TNF-α and IL-1α Stimulate Neuropeptide Gene Expression in Adrenochromaffin Cells

Proinflammatory Cytokines TNFand IL-1 Stimulate Neuropeptide Gene Expression in Adrenochromaffin Cells V. TURQUIERa, H. VAUDRYa, L. YONa, C-M. HSUb, D. AIT-ALIa, L. GRUMOLATOa, L. E. EIDENb and Y. ANOUARa aEuropean Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France bSection on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892, USA