Johann Guillemot

Biochemical Characterization and Immunocytochemical Localization of EM66, a Novel Peptide Derived from Secretogranin II, in the Rat Pituitary and Adrenal Glands

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine 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.

Development of Novel Tools for the Diagnosis and Prognosis of Pheochromocytoma Using Peptide Marker Immunoassay and Gene Expression Profiling Approaches

Abstract:  Pheochromocytomas (PHEOs) are rare catecholamine‐producing neoplasias that arise from chromaffin cells of the adrenal medulla or from extra‐adrenal locations. These neuroendocrine tumors are usually benign, but may also present as or develop into a malignancy. There are currently no means to predict or to cure malignant tumors which have a poor prognosis. We have recently validated several assays for the measurement of peptides derived from chromogranin A (CgA) and secretogranin II (SgII) in order to determine whether these secreted neuroendocrine products could provide useful, complementary markers for the diagnosis and prognosis of PHEOs. Both the CgA‐derived peptide WE14 and the SgII‐derived peptide EM66 proved to be sensitive circulating markers for the diagnosis of PHEO. In addition, much higher EM66 levels were measured in benign than in malignant tumoral tissues, suggesting that this peptide could represent a valuable tool for the prognosis of PHEO. We have also initiated a comparative microarray study of benign and malignant PHEOs, which allowed the identification of a set of about 100 genes that were differentially expressed and best discriminated the two types of tumors. A large majority of these genes were expressed at lower levels in the malignant disease and were associated with various characteristics of chromaffin cells, such as hormone secretion signaling and machinery, peptide maturation, and cellular morphology. Altogether, these studies provide novel tools for the management of PHEO, and new insights for the understanding of tumorigenesis in chromaffin cells, which may offer potential therapeutic strategies.

IGF2 Promotes Growth of Adrenocortical Carcinoma Cells, but Its Overexpression Does Not Modify Phenotypic and Molecular Features of Adrenocortical Carcinoma

Insulin-like growth factor 2 (IGF2) overexpression is an important molecular marker of adrenocortical carcinoma (ACC), which is a rare but devastating endocrine cancer. It is not clear whether IGF2 overexpression modifies the biology and growth of this cancer, thus more studies are required before IGF2 can be considered as a major therapeutic target. We compared the phenotypical, clinical, biological, and molecular characteristics of ACC with or without the overexpression of IGF2, to address these issues. We also carried out a similar analysis in an ACC cell line (H295R) in which IGF2 expression was knocked down with si- or shRNA. We found no significant differences in the clinical, biological and molecular (transcriptomic) traits between IGF2-high and IGF2-low ACC. The absence of IGF2 overexpression had little influence on the activation of tyrosine kinase pathways both in tumors and in H295 cells that express low levels of IGF2. In IGF2-low tumors, other growth factors (FGF9, PDGFA) are more expressed than in IGF2-high tumors, suggesting that they play a compensatory role in tumor progression. In addition, IGF2 knock-down in H295R cells substantially impaired growth (>50% inhibition), blocked cells in G1 phase, and promoted apoptosis (>2-fold). Finally, analysis of the 11p15 locus showed a paternal uniparental disomy in both IGF2-high and IGF2-low tumors, but low IGF2 expression could be explained in most IGF2-low ACC by an additional epigenetic modification at the 11p15 locus. Altogether, these observations confirm the active role of IGF2 in adrenocortical tumor growth, but also suggest that other growth promoting pathways may be involved in a subset of ACC with low IGF2 expression, which creates opportunities for the use of other targeted therapies.

Biochemical Characterisation and Immunohistochemical Localisation of the Secretogranin II‐Derived Peptide EM66 in the Hypothalamus of the Jerboa (Jaculus orientalis): Modulation by Food Deprivation

The neuroendocrine protein secretogranin II is the precursor of several neuropeptides, including secretoneurin and a novel 66‐amino acid peptide, EM66, the sequence of which has been highly conserved across the vertebrae phylum. The presence of EM66 has been detected in the adult and fetal human adrenal gland, as well as the rat pituitary and adrenal glands. The present study aimed to explore a possible neuroendocrine role of EM66 by analysing its occurrence and distribution within the jerboa hypothalamus, and its potential implication in the control of feeding behaviour. High‐performance liquid chromatography analysis of jerboa hypothalamic extracts combined with a radioimmunoassay of EM66 revealed a single peak of immunoreactive material exhibiting the same retention time as recombinant EM66. Immunocytochemical labelling showed that EM66‐producing neurones are widely distributed in several hypothalamic regions, including the preoptic area, the suprachiasmatic, supraoptic, parvocellular paraventricular and arcuate nuclei, and the lateral hypothalamus. Food deprivation for 5 days induced a significant increase in the number of EM66‐containing neurones within the arcuate nucleus (105% increase) and the parvocellular aspect of the paraventricular nucleus (115% increase), suggesting that EM66 could be involved in the control of feeding behaviour and/or the response to stress associated with fasting. Altogether, these data reveal the physiological plasticity of the EM66 system in the hypothalamus and implicate this novel peptide in the regulation of neuroendocrine functions.

Granins and their derived peptides in normal and tumoral chromaffin tissue: Implications for the diagnosis and prognosis of pheochromocytoma

Pheochromocytomas are rare catecholamine-secreting tumors that arise from chromaffin tissue within the adrenal medulla and extra-adrenal sites. Typical clinical manifestations are sustained or paroxysmal hypertension, severe headaches, palpitations and sweating resulting from hormone excess. However, their presentation is highly variable and can mimic many other diseases. The diagnosis of pheochromocytomas depends mainly upon the demonstration of catecholamine excess by 24-h urinary catecholamines and metanephrines or plasma metanephrines. Occurrence of malignant pheochromocytomas can only be asserted by imaging of metastatic lesions, which are associated with a poor survival rate. The characterization of tissue, circulating or genetic markers is therefore crucial for the management of these tumors. Proteins of the granin family and their derived peptides are present in dense-core secretory vesicles and secreted into the bloodstream, making them useful markers for the identification of neuroendocrine cells and neoplasms. In this context, we will focus here on reviewing the distribution and characterization of granins and their processing products in normal and tumoral chromaffin cells, and their clinical usefulness for the diagnosis and prognosis of pheochromocytomas. It appears that, except SgIII, all members of the granin family i.e. CgA, CgB, SgII, SgIV-SgVII and proSAAS, and most of their derived peptides are present in adrenomedullary chromaffin cells and in pheochromocytes. Moreover, besides the routinely used CgA test assays, other assays have been developed to measure concentrations of tissue and/or circulating granins or their derived peptides in order to detect the occurrence of pheochromocytomas. In most cases, elevated levels of these entities were found, in correlation with tumor occurrence, while rarely discriminating between benign and malignant neoplasms. Nevertheless, measurement of the levels of granins and derived peptides improves the diagnostic sensitivity and may therefore provide a complementary tool for the management of pheochromocytomas. However, the existing data need to be substantiated in larger groups of patients, particularly in the case of malignant disease.

Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas.

Abstract:  The aim of the present study was to compare the expression levels of secretogranin II (SgII), prohormone convertases (PC)1 and PC2, and the proteolytic processing of SgII in benign versus malignant pheochromocytomas. Quantitative (Q)‐PCR experiments indicated that SgII, PC1, and PC2 mRNAs were overexpressed in pheochromocytoma compared to non‐tumoral chromaffin cells (P < 0.001) and in benign compared to malignant tumors (P < 0.01). Western blot analysis using a human SgII antiserum revealed the occurrence of a 97‐kDa band corresponding to the expected size of SgII, with significantly higher quantities in benign than in malignant tumors (P < 0.05). Using antisera directed against sequential regions of SgII (N‐terminal, secretoneurin [SN], EM66, internal, and C‐terminal sequences), we observed distinct processing profiles between benign and malignant pheochromocytomas. In contrast, using PC1 and PC2 antisera no differences between the two types of tumors were found. RIA measurement showed that EM66 median values between benign and malignant chromaffin cell tumors were significantly different (128.5 vs. 6.3 ng/mg protein, respectively; P < 0.001). Taken together, these results indicate that, in pheochromocytoma, malignancy is associated with reduced PC1, PC2, and SgII mRNA expression and decreased levels of processing products of SgII, in line with the low concentrations of EM66 that occur in malignant tumors. These data support the notion that SgII‐processing products, such as EM66, could represent prognostic markers of pheochromocytomas.

Metoclopramide stimulates catecholamine- and granin-derived peptide secretion from pheochromocytoma cells through activation of serotonin type 4 (5-HT4) receptors

UNLABELLED The gastroprokinetic agent metoclopramide is known to stimulate catecholamine secretion from pheochromocytomas. The aim of the study was to investigate the mechanism of action of metoclopramide and expression of serotonin type 4 (5-HT(4)) receptors in pheochromocytoma tissues. Tissue explants, obtained from 18 pheochromocytomas including the tumor removed from a 46-year-old female patient who experienced life-threatening hypertension crisis after metoclopramide administration and 17 additional pheochromocytomas (9 benign and 8 malignant) were studied. Cultured pheochromocytoma cells derived from the patient who previously received metoclopramide were incubated with metoclopramide and various 5-HT(4) receptor ligands. In addition, total mRNAs were extracted from all the 18 tumors. Catecholamine- and granin-derived peptide concentrations were measured in pheochromocytoma cell incubation medium by HPLC and radioimmunological assays. In addition, expression of 5-HT(4) receptor mRNAs in the 18 pheochromocytomas was investigated by the use of reverse transcriptase-PCR. RESULTS Metoclopramide and the 5-HT(4) receptor agonist cisapride were found to activate catecholamine- and granin-derived peptide secretions by cultured tumor cells. Metoclopramide- and cisapride-evoked catecholamine- and granin-derived peptide productions were inhibited by the 5-HT(4) receptor antagonist GR 113808. 5-HT(4) receptor mRNAs were detected in the patients tumor and the series of 17 additional pheochromocytomas. This study shows that pheochromocytomas express functional 5-HT(4) receptors that are responsible for the stimulatory action of metoclopramide on catecholamine- and granin-derived peptide secretion. All 5-HT(4) receptor agonists must therefore be contraindicated in patients with proven or suspected pheochromocytoma.

Immunohistochemical distribution of the secretogranin II-derived peptide EM66 in the rat hypothalamus: A comparative study with jerboa

EM66 is a 66-amino acid peptide derived from secretogranin II, a member of granin acidic secretory protein family, by proteolytic processing. EM66 has been previously characterized in the jerboa (Jaculus orientalis) hypothalamus and its potential implication in the neuroendocrine regulation of feeding behaviour has been demonstrated. In the present study, an immunohistochemical analysis of the localization of EM66 within hypothalamic structures of rat was performed and compared to the distribution of EM66 in the jerboa hypothalamus. In the rat hypothalamus, as in the jerboa, EM66 immunostaining was detected in the parvocellular paraventricular, preoptic and arcuate nuclei, as well as the lateral hypothalamus which displayed an important density of EM66-producing neurones. However, unlike jerboa, the suprachiasmatic and supraoptic nuclei of the rat hypothalamus were devoid of cellular EM66-immunolabeling. Thus, the novel peptide EM66 may exert common neuroendocrine activities in rat and jerboa, e.g. control of food intake, and species-specific roles in jerboa such as the regulation of biological rhythms and hydromineral homeostasis. These results suggest the existence of differences between jerboas and rats in neuroendocrine regulatory mechanisms involving EM66.

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.