Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs.

Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs

Summary Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Gene expression patterns of pro-opiomelanocortin-processing enzymes PC1 and PC2 during postnatal development of rat corticotrophs.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the anterior pituitary cells of developing rats by a double staining procedure using in situ RT-PCR and an immunofluorescence technique. In the adult, both PC1 mRNA and PC2 mRNA were expressed in corticotrophs, gonadotrophs, thyrotrophs, and mammotrophs. These cells, except for corticotrophs, had previously been considered to be ones in which proprotein processing does not take place, but both PC1 and PC2 may be necessary to process other proteins, such as granin family proteins, having proteolytic cleavage sites and located in secretory granules of the above trophs. In addition, no PC1 or PC2 mRNA was expressed in somatotrophs, which is consistent with the fact that somatotrophs do not contain these granins. In addition, 7B2 mRNA was expressed in these PC2-positive trophs, suggesting that there is a functional relationship between PC2 and 7B2 proteins. We found that alpha-MSH was expressed in the corticotrophs of the postnatal rat and that the number of alpha-MSH-immunopositive corticotrophs decreased as development proceeded. Because the changes in the pattern of POMC processing are considered to depend on the relative expression levels of PC1 and PC2, PC1 and PC2 mRNAs were examined in corticotrophs during postnatal development. We found a decrease in the number of PC2 mRNA-positive cells, which coincided with one in the number of alpha-MSH-immunopositive corticotrophs, as postnatal development proceeded. Our present data demonstrate that the alpha-MSH production varies directly in accordance with the expression of PC2. We also discuss the possible significance of alpha-MSH production during the postnatal period.     

The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects

The chromogranins A (CgA) and B (CgB) and secretogranin II (SgII) constitute the main members of a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These genetically distinct proteins, CgA, CgB, SgII and the less well known secretogranins III-VII are collectively referred to as 'granins' and characterised by numerous pairs of basic amino acids as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC 1/3 and PC2. This review is directed towards comparative and functional aspects of the granins with emphasis on their phylogenetically conserved sequences. Recent developments provide ample evidence of widely different effects and targets for the intact granins and their derived peptides, intracellularly in the directed trafficking of storage components during granule maturation and extracellularly in autocrine, paracrine and endocrine interactions. Most of the effects assigned to the granin derived peptides fit into patterns of direct or indirect inhibitory modulations of major functions. So far, peptides derived from CgA (vasostatins, chromacin, pancreastatin, WE-14, catestatin and parastatin), CgB (secretolytin) and SgII (secretoneurin) are the most likely candidates for granin-derived regulatory peptides, of postulated relevance not only for homeostatic processes, but also for tissue assembly and repair, inflammatory responses and the first line of defence against invading microorganisms.     

Regulation of regional expression in rat brain PC2 by thyroid hormone/characterization of novel negative thyroid hormone response elements in the PC2 promoter

The prohormone convertases (PCs) PC1 and PC2 are involved in the tissue-specific endoproteolytic processing of neuropeptide precursors within the secretory pathway. We previously showed that changes in thyroid status altered pituitary PC2 mRNA and that this regulation was due to triiodothyronine-dependent interaction of the thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large proximal region of the human PC2 promoter. In the current study, we examined the in vivo regulation of brain PC2 mRNA by thyroid status and found that 6-n-propyl-2-thiouracil-induced hypothyroidism stimulated, whereas thyroxine-induced hyperthyroidism suppressed, PC2 mRNA levels in the rat hypothalamus and cerebral cortex. To address the mechanism of T3 regulation of the PC2 gene, we used human PC2 (hPC2) promoter constructs transiently transfected into GH3 cells and found that triiodothyronine negatively and 9-cis-retinoic acid positively regulated hPC2 promoter activity. EMSAs, using purified TRalpha1 and retinoid X receptor-beta (RXRbeta) proteins demonstrated that TRalpha bound the distal putative nTRE-containing oligonucleotide in the PC2 promoter, and RXR bound to both nTRE-containing oligonucleotides. EMSAs with oligonucleotides containing deletion mutations of the nTREs demonstrated that the binding to TR and RXR separately is reduced, but specific binding to TR and RXR together persists even with deletion of each putative nTRE. We conclude that there are two novel TRE-like sequences in the hPC2 promoter and that these regions act in concert in a unique manner to facilitate the effects of thyroid hormone and 9-cis-retinoic acid on PC2.     

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.     

The Neuroendocrine Proteins Secretogranin II and III Are Regionally Conserved and Coordinately Expressed with Proopiomelanocortin in Xenopus Intermediate Pituitary

Abstract: Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretory granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from a Xenopus intermediate pituitary cDNA library. A comparative analysis of the Xenopus and mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in the trans -Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. When Xenopus intermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15- and 35-fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30-fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin family.     

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.     

Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA-mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.     

Proteolytic processing of chromogranin A by the prohormone convertase PC2

The neuroendocrine secretory protein chromogranin A (CgA) is a precursor for various biologically active peptides. Several single and paired basic residues are present within its primary amino acid sequence comprising cleavage sites for prohormone convertases. In this study, SH-SY5Y human neuroblastoma cells were stably transfected with the prohormone convertase PC2 to analyse the proteolytic processing of endogenous chromogranin A and, in particular, the formation of the chromogranin-A-derived peptide GE-25. Our analyses revealed a significant change in the pattern of proteolytic conversion of chromogranin A in cells expressing PC2. Mock-transfected control cells contained mainly the intact chromogranin A molecule and hardly any shorter products were found. On the other hand, PC2-transfected cells showed extensive processing of chromogranin A, resulting in significantly lower amounts of the intact precursor and especially high levels of the free peptide GE-25.     

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.     

Immunological characterization of the endoproteases PC1 and PC2 in adrenal chromaffin granules and in the pituitary gland.

Specific antisera against synthetic fragment of the endoproteases, PC1 and PC2, were used to characterize these proteins. In one-dimensional immunoblots these antisera labelled components of 85 kDa for PC1 and of 70 kDa for PC2 in purified bovine chromaffin granules and anterior and posterior pituitary of ox and rat. In membranes of bovine chromaffin granules glycoprotein H was identified as the major PC2 immunoreactive spot. A major part of these endoproteases appeared membrane bound.     

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.     

Molecular cloning and cellular expression of crustacean PC2-like prohormone convertase

PC2 prohormone convertases are enzymes involved in the proteolytic maturation of neuropeptide precursors. In the present work, a cDNA encoding a PC2-like enzyme (OrlPC2) was cloned from crayfish eyestalk ganglia (medulla terminalis) containing the X-organ, a major neuroendocrine center. The predicted 634 amino acid preproprotein exhibits highest sequence identity, especially in the catalytic domain, with PC2s from arthropods and nematodes, and less with mollusc and vertebrate enzymes. It was demonstrated by in situ hybridization on crayfish medulla terminalis sections that OrlPC2 is expressed in a large number of neuron perikarya, including those producing the well known crustacean hyperglycemic hormone.     

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi- intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.     

Cleavage of recombinant proenkephalin and blockade mutants by prohormone convertases 1 and 2: an in vitro specificity study

Proenkephalin (PE) derived-peptides are thought to be generated predominantly through endoproteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). In order to compare cleavage site preferences of these convertases, we studied the processing of recombinant wild-type rat PE and of two mutant PEs by recombinant purified mouse PC1 and PC2. Western blot analyses of timed digestions showed that both mouse PC1 and PC2 were able to produce a variety of large and intermediate sized-peptides from wild-type PE as well as from the precursors mutated at initial blockade sites. PC2 exhibited a broader specificity against PE than PC1, generating a much greater number of peptide products. Mass spectrometric identification of cleavage products showed that PC2 appeared to be the principal enzyme involved in the generation of smaller active opioids. Both enzymes were able to cleave various KR- and KK-containing sites, but PC2 was also able to cleave efficiently at an RR-V site and a KK-M site not cleaved by PC1, suggesting the exclusion of large aliphatic residues at the P1' position in PC1 cleavage. Alternative cleavage sites were readily chosen by convertases in blockade mutants, confirming in vivo results that cleavages do not follow an obligatory order. Furthermore, glycosylated PE was less efficiently processed by PC2, indicating that glycosylation may serve as a mechanism to hinder processing.     

Defective prodynorphin processing in mice lacking prohormone convertase PC2

Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.     

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.     

Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.