Characterization of the EM66 Biomarker in the Pituitary and Plasma of Healthy Subjects With Different Gonadotroph Status and Patients With Gonadotroph Tumor.

Granins and their derived-peptides are useful markers of secretion from normal and tumoral neuroendocrine cells. The need to identify new diagnostic markers for neuroendocrine tumors, including pituitary tumors prompted us to determine plasma levels of the secretogranin II-derived peptide EM66 in healthy volunteers with different gonadotroph status and to evaluate its usefulness as a circulating marker for the diagnosis of gonadotroph tumor. Using a radioimmunoassay, we determined plasma EM66 concentrations in healthy men and women volunteers in different physiological conditions in relation with the gonadotroph function. Our results revealed that in men, in women with or without contraception, in pregnant or post-menopausal women, plasma EM66 concentrations are not significantly different, and did not show any correlation with gonadotropin levels. In addition, stimulation or inhibition tests of the gonadotroph axis had no effect on EM66 levels, whatever the group of healthy volunteers investigated while gonadotropin levels showed the expected variations. Immunohistochemical experiments and HPLC analysis showed the occurrence of EM66 in pituitary gonadotroph, lactotroph and corticotroph tumors but not in somatotroph tumor. In patients with gonadotroph or lactotroph tumor, plasma EM66 levels were 1.48 (0.82-4.38) ng/ml and 2.49 (1.19-3.54) ng/ml, respectively. While median value of EM66 was significantly lower in patients with gonadotroph tumor compared to healthy volunteers [2.59 (0.62-4.95) ng/ml], plasma EM66 concentrations were in the same range as normal values and did not show any correlation with gonadotropin levels. These results show that plasma EM66 levels are independent of the activity of the gonadotroph axis in healthy volunteers and, while EM66 levels are reduced in gonadotroph tumors, plasma EM66 does not provide a helpful marker for the diagnosis of these tumors.

Characterization and plasma measurement of the WE-14 peptide in patients with pheochromocytoma.

Granins and their derived peptides are valuable circulating biological markers of neuroendocrine tumors. The aim of the present study was to investigate the tumoral chromogranin A (CgA)-derived peptide WE-14 and the potential advantage to combine plasma WE-14 detection with the EM66 assay and the existing current CgA assay for the diagnosis of pheochromocytoma. Compared to healthy volunteers, plasma WE-14 levels were 5.4-fold higher in patients with pheochromocytoma, but returned to normal values after surgical resection of the tumor. Determination of plasma CgA and EM66 concentrations in the same group of patients revealed that the test assays for these markers had an overall 84% diagnostic sensitivity, which is identical to that determined for WE-14. However, we found that WE-14 measurement improved the diagnostic sensitivity when combined with the results of CgA or EM66 assays. By combining the results of the three assays, the sensitivity for the diagnosis of pheochromocytoma was increased to 95%. In fact, the combination of WE-14 with either CgA or EM66 test assays achieved 100% sensitivity for the diagnosis of paragangliomas and sporadic or malignant pheochromocytomas if taken separately to account for the heterogeneity of the tumor. These data indicate that WE-14 is produced in pheochromocytoma and secreted into the general circulation, and that elevated plasma WE-14 levels are correlated with the occurrence of this chromaffin cell tumor. In addition, in association with other biological markers, such as CgA and/or EM66, WE-14 measurement systematically improves the diagnostic sensitivity for pheochromocytoma. These findings support the notion that granin-processing products may represent complementary tools for the diagnosis of neuroendocrine tumors.

Immunocytochemical distribution of EM66 within the hypothalamic parvocellular paraventricular nucleus: colocalization with CRH and TRH but no plasticity related to acute stress and thyroidectomy in the rat.

EM66 is a secretogranin II-derived peptide strongly expressed within hypothalamic neuroendocrine areas such as the parvocellular aspect of the paraventricular nucleus (pPVN) as well as the median eminence (ME), suggesting a hypophysiotropic role for this neuropeptide. The aim of the present study was to explore such a role in the corticotrope and thyrotrope axes. We analyzed EM66 occurrence respectively in CRH and TRH neurosecretory cells of the rat pPVN by double immunohistochemistry. Functionally, we studied the effect of acute stress (immobilization for 2 h or cold exposure at 5°C for 4 h) and hypothyroidism (induced by 1-week thyroidectomy) on EM66 immunoreactivity (IR) within the pPVN. Double immunohistochemical labeling revealed that EM66-IR colocalized with CRH or TRH labelings within pPVN hypophysiotropic neurons as well as the axon terminals of the external layer of the ME. Because TRH neuronal population of the pPVN is completely distinct from the CRH neurosecretory system, our data demonstrate the existence of at least two distinct EM66 neuronal populations in the rat pPVN. Acute immobilization or cold exposure stresses did not affect EM66 expression as evaluated by the number of EM66-IR neurons within the pPVN. These results suggest that EM66 does not participate to the phenotypic plasticity of hypothalamic parvocellular neurons in response to acute stress. In addition, short-term hypothyroidism did not provoke any significant variation of the number of intraparaventricular EM66 neurons, indicating that EM66 expression would be insensitive to short-term hypothyroidism despite its occurrence within TRH neurons. Thus, the present data show the occurrence of EM66 in distinct areas of the rat PVN but its expression is not coregulated with those of CRH and TRH during acute stress and hypothyroidism.

EM66-containing neurones in the hypothalamic parvicellular paraventricular nucleus of the rat: no plasticity related to acute immune stress.

OBJECTIVES AND METHODS: Neuropeptides, as the main neuroendocrine system effectors, regulate notably the response to different stressors via a secretory plasticity within their respective hypothalamic neuronal populations. The aim of the present study was to explore by immunocytochemistry the occurrence and the potential expression plasticity of the novel neuropeptide EM66 in the CRH neurones of stressed rats. RESULTS: The secretogranin II (SgII)-derived peptide EM66 is strongly expressed within hypothalamic neuroendocrine areas such as the parvocellular aspect of the paraventricular nucleus (pPVN) as well as the median eminence, suggesting a probable hypophysiotropic effect of this peptide. As a first approach to investigate such a role, we evaluated by immunohistochemistry EM66 expression within the pPVN following acute immune stress induced by lipopolysaccharide (LPS) or interleukin-1ß (IL-1ß) injection in rat. This study showed that EM66 is present in the pPVN but the number of EM66 immunolabeled cells did not fluctuate in this structure following LPS peripheral injection. In line with this observation, an intracerebroventricular injection of IL-1ß did not provoke any significant variation of the number of intraparaventricular EM66 neurones. CONCLUSION: The present data revealed for the first time that EM66 expression would be insensitive to the central and peripheral cytokines within the neurosecretory hypothalamic pPVN. This result indicates that EM66 does not participate to the phenotypic plasticity of hypothalamic parvicellular neurones in response to acute inflammatory stress.

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 of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival.

Pheochromocytomas are catecholamine-producing tumors which are generally benign, but which can also present as or develop into malignancy. Molecular pathways of malignant transformation remain poorly understood. Pheochromocytomas express various trophic peptides which may influence tumoral cell behavior. Here, we investigated the expression of trophic amidated peptides, including pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), and adrenomedullin (AM), and their receptors in benign and malignant pheochromocytomas in order to assess their potential role in chromaffin cell tumorigenesis and malignant transformation. PACAP, NPY, and AM are expressed in the majority of pheochromocytomas studied; NPY exhibiting the highest mRNA levels relative to reference genes. Although median gene expression or peptide levels were systematically lower in malignant compared to benign tumors, no statistically significant difference was found. Among all the receptors of these peptides that were analyzed, only the AM receptor RDC1 displayed a differential expression between benign and malignant pheochromocytomas. This receptor exhibited a fourfold higher expression in malignant than in benign tumors. AM and stromal cell-derived factor 1, which has also been described as a ligand for RDC1, increased the number of human pheochromocytoma cells in primary culture and exerted anti-apoptotic activity on rat pheochromocytoma PC12 cells. In addition, RDC1 gene silencing decreased the number of viable PC12 cells. This study shows the expression of several trophic peptides and their receptors in benign and malignant pheochromocytomas, and suggests that AM and its RDC1 receptor could be involved in chromaffin cell tumorigenesis through pro-survival effects. Therefore, AM and RDC1 may represent valuable targets for the treatment of malignant pheochromocytomas.