Identification of a chromogranin A domain that mediates binding to secretogranin III and targeting to secretory granules in pituitary cells and pancreatic beta-cells

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca(2+)-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48-111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214-373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.     

Secretogranin III binds to cholesterol in the secretory granule membrane as an adapter for chromogranin A

Granin-family proteins, including chromogranin A (CgA) and secretogranin III (SgIII), are transported to secretory granules (SGs) in neuroendocrine cells. We previously showed that SgIII binds strongly to CgA in an intragranular milieu and targets CgA to SGs in pituitary and pancreatic endocrine cells. In this study, we demonstrated that with a sucrose density gradient of rat insulinoma-derived INS-1 cell homogenates, SgIII was localized to the SG fraction and was fractionated to the SG membrane (SGM) despite lacking the transmembrane region. With depletion of cholesterol from the SGM using methyl-beta-cyclodextrin, SgIII was impaired to bind to the SGM. Both SgIII and CgA were solubilized from the SGM by Triton X-100 in contrast to the Triton X-100 insolubility of carboxypeptidase E. SgIII and carboxypeptidase E strongly bound to the SGM-type liposome in intragranular conditions, but CgA did not. Instead, CgA bound to the SGM-type liposome only in the presence of SgIII. Immunocytochemical and pulse-chase experiments revealed that SgIII deleting the N-terminal lipid-binding region missorted to the constitutive pathway in mouse corticotroph-derived AtT-20 cells. Thus, we suggest that SgIII directly binds to cholesterol components of the SGM and targets CgA to SGs in pituitary and pancreatic endocrine cells.     

Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system:     

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.     

Immunohistochemical and Biochemical Studies with Region-Specific Antibodies to Chromogranins A and B and Secretogranins II and III in Neuroendocrine Tumors

This short review deals with our investigations in neuroendocrine tumors (NETs) with antibodies against defined epitopes of chromogranins (Cgs) A and B and secretogranins (Sgs) II and III. The immunohistochemical expression of different epitopes of the granin family of proteins varies in NE cells in normal human endocrine and non-endocrine organs and in NETs, suggesting post-translational processing. In most NETs one or more epitopes of the granins were lacking, but variations in the expression pattern occurred both in benign and malignant NETs. A few epitopes displayed patterns that may be valuable in differentiating between benign and malignant NET types, e.g., well-differentiated NET types expressed more CgA epitopes than the poorly differentiated ones and C-terminal secretoneurin visualized a cell type related to malignancy in pheochromocytomas. Plasma concentrations of different epitopes of CgA and CgB varied. In patients suffering from carcinoid tumors or endocrine pancreatic tumors the highest concentrations were found with epitopes from the mid-portion of CgA. For CgB the highest plasma concentrations were recorded for the epitope 439–451. Measurements of SgII showed that patients with endocrine pancreatic tumors had higher concentrations than patients with carcinoid tumors or pheochromocytomas. SgIII was not detectable in patients with NETs.     

Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours

INTRODUCTION: Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. MATERIALS AND METHODS: Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. RESULTS: Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154-165 (N-terminal secretoneurin), the SgII 172-186 (C-terminal Secretoneurin) and the SgII 225-242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. CONCLUSION: Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.     

Multiple Sorting Systems for Secretory Granules Ensure the Regulated Secretion of Peptide Hormones

Prior to secretion, regulated peptide hormones are selectively sorted to secretory granules (SGs) at the trans‐Golgi network (TGN) in endocrine cells. Secretogranin III (SgIII) appears to facilitate SG sorting process by tethering of protein aggregates containing chromogranin A (CgA) and peptide hormones to the cholesterol‐rich SG membrane (SGM). Here, we evaluated the role of SgIII in SG sorting in AtT‐20 cells transfected with small interfering RNA targeting SgIII. In the SgIII‐knockdown cells, the intracellular retention of CgA was greatly impaired, and only a trace amount of CgA was localized within the vacuoles formed in the TGN, confirming the significance of SgIII in both the tethering of CgA‐containing aggregates and the establishment of the proper SG morphology. Although the intracellular retention of proopiomelanocortin (POMC) was considerably impaired in SgIII‐knockdown cells, residual adrenocorticotropic hormone (ACTH)/POMC was still localized to some few remaining SGs together with another granin protein, secretogranin II (SgII), and was secreted in a regulated manner. Biochemical analyses indicated that SgII bound directly to the SGM in a cholesterol‐dependent manner and was able to retain the aggregated form of POMC, revealing a latent redundancy in the SG sorting and retention mechanisms, that ensures the regulated secretion of bioactive peptides.     

Secretogranin III directs secretory vesicle biogenesis in mast cells in a manner dependent upon interaction with chromogranin A

Mast cells are granular immunocytes that reside in the body's barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the proteins that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) protein is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1-210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1.     

Immunohistochemical localization of huntingtin-associated protein 1 in endocrine system of the rat.

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.     

Chromogranin A is present in and released by fish endocrine tissue

Chromogranin A (CgA) is a protein that is present in many mammalian endocrine cells and co-secreted with their resident hormones. We have demonstrated the presence of CgA by immunohistology in the ultimobranchial glands and corpuscles of Stannius of rainbow trout. CgA was also detected by radioimmunoassay in the medium of incubated coho salmon ultimobranchial glands. Our observations demonstrate the presence of CgA in endocrine glands of evolutionarily divergent species. These observations are consistent with the hypothesis that CgA participates in the secretory process of a wide variety of hormones.     

Targeted ablation of the chromogranin a (Chga) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins

Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.     

Identification of a novel pituitary-specific chicken gonadotropin-releasing hormone receptor and its splice variants

In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%-65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.     

Immunoreactivities for chromogranin A and B, and secretogranin II in the guinea-pig endocrine pancreas

The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

Immunoreactivities for chromogranin A and B,and secretogranin II in the guinea-pig endocrine pancreas

Summary The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

PD-L1 Expression in Normal Endocrine Tissues Is Not Increased Despite High Incidence of PD-1 Inhibitor-Associated Endocrinopathies

ObjectiveTreatment with immune-checkpoint inhibitors often results in endocrine immune-related adverse events (irAEs), affecting the pituitary, thyroid, adrenal, and parathyroid glands and pancreas. The mechanism underlying the endocrine irAEs has not been fully elucidated, and it remains unclear why endocrine organs are so commonly affected. In the present study, we evaluated immunostaining patterns of programmed death-ligand 1 (PD-L1) in normal endocrine tissues to determine whether increased expression may explain the predilection of endocrinopathies in patients treated with programmed cell death-1 inhibitors.MethodsNormal formalin-fixed paraffin-embedded endocrine tissues (pituitary, thyroid, adrenal, pancreas, and parathyroid) were collected from our hospital’s pathology tissue archive. The tissues were assessed for membranous and cytoplasmic PD-L1 immunostaining using the Dako 22C3 pharmDx assay on an automated staining platform.ResultsWe examined 49 endocrine tissues, including 12 thyroid, 5 pancreatic, 17 adrenal, 5 parathyroid, and 10 pituitary samples. Samples with less than 1% membranous PD-L1–positive cells were considered negative, while those with more than 1% of PD-L1 membranous staining were considered positive. Immunostaining result of immune-related cells was also evaluated, considering the cytoplasmic PD-L1–positive cells with the same cutoff of 1%. None of the endocrine tissues demonstrated PD-L1 positivity higher than 1% in the relevant cells.ConclusionWhile our results do not suggest a role of PD-L1 expression in the pathogenesis of endocrine irAEs, they may serve as a basis for future studies further investigating the mechanisms of autoimmune, inflammatory, or malignant endocrine conditions.     

Different ghrelin localisation in adult human and rat endocrine pancreas

Ghrelin is an endocrine peptide that has been identified in gastric oxyntic glands and that induces growth hormone secretion in the pituitary gland. This growth hormone secretagogue is expressed in many tissues such as stomach, pituitary gland, thyroid, testis, placenta and pancreas. Initial studies of ghrelin focused on its role as a circulating orexigenic signal. However, ghrelin has also been found to be involved in the modulation of glucose homeostasis. Although a number of studies have reported ghrelin expression in developing pancreas, the location of ghrelin-immunoreactive cells in adult pancreas (epsilon cells) remains controversial. In this study, we have analysed the distribution of pancreatic epsilon cells in adult human and rat islets by immunohistochemistry and in situ hybridisation. In humans, our immunohistochemical analysis has shown that ghrelin is expressed in glucagon-secreting cells, whereas in rats, it is present in insulin-secreting cells. Similar observations have been revealed by in situ hybridisation.     

The fringe molecules induce endocrine differentiation in embryonic endoderm by activating cMyt1/cMyt3

Endocrine differentiation in the early embryonic pancreas is regulated by Notch signaling. Activated Notch signaling maintains pancreatic progenitor cells in an undifferentiated state, whereas suppression of Notch leads to endocrine cell differentiation. Yet it is not known what mechanism is employed to inactivate Notch in a correct number of precursor cells to balance progenitor proliferation and differentiation. We report that an established Notch modifier, Manic Fringe (Mfng), is expressed in the putative endocrine progenitors, but not in exocrine pancreatic tissues, during early islet differentiation. Using chicken embryonic endoderm as an assaying system, we found that ectopic Mfng expression is sufficient to induce endodermal cells to differentiate towards an endocrine fate. This endocrine-inducing activity depends on inactivation of Notch. Furthermore, ectopic Mfng expression induces the expression of basic helix-loop-helix gene, Ngn3, and two zinc finger genes, cMyt1 and cMyt3. These results suggest that Mfng-mediated repression of Notch signaling could serve as a trigger for endocrine islet differentiation.