030320 the Chromogranin–Secretogranin Family

The new england journal of medicine

review article

mechanisms of disease The Chromogranin–Secretogranin Family

Laurent Taupenot, Ph.D., Kimberly L. Harper, M.D., and Daniel T. O’Connor, M.D.

From the Department of Medicine (L.T., eurons and neuroendocrine cells contain membrane- K.L.H., D.T.O.) and the Center for Molecular delimited pools of peptide hormones, biogenic amines, and neurotransmit- Genetics (D.T.O.), University of California n at San Diego, La Jolla; and the Veterans ters with a characteristic electron-dense appearance on transmission electron Affairs San Diego Healthcare System, San microscopy (Fig. 1). These vesicles, which are present throughout the neuroendocrine Diego, Calif. (L.T., K.L.H., D.T.O.). Address system1,2 and in a variety of neurons, store and release chromogranins and secretogranins reprint requests to Dr. O’Connor at the 3,4 Department of Medicine (9111H), Univer- (also known as “granins”), a unique group of acidic, soluble secretory proteins. The sity of California at San Diego, 3350 La Jolla three “classic” granins are chromogranin A, which was first isolated from chromaffin Village Dr., San Diego, CA 92161, or at cells of the adrenal medulla5,6; chromogranin B, initially characterized in a rat pheochro- [email protected]. mocytoma cell line7; and secretogranin II (sometimes called chromogranin C), which 8,9 N Engl J Med 2003;348:1134-49. was originally described in the anterior pituitary. Four other acidic secretory pro- Copyright © 2003 Massachusetts Medical Society. teins were later proposed for membership in the granin family10: secretogranin III (or 1B1075),11 secretogranin IV (or HISL-19),12 secretogranin V (or 7B2),13 and secretogranin VI (or NESP55).14 In this article, we review aspects of the structures, biochemical properties, and clinical importance of granins, with particular emphasis on chromogranin A, the granin that was discovered first and that has been studied most extensively. We discuss how granins contribute to the formation of secretory granules and how, as prohormones, they give rise to bioactive peptides through proteolytic processing. Because of their ubiquitous distribution in neuroendocrine and nervous-system tissues and their cose- cretion with resident peptide hormones and biogenic amines, granins are valuable in- dicators of sympathoadrenal activity and clinically useful markers of secretion from normal and neoplastic neuroendocrine cells.1,15-17 Indeed, numerous studies have documented the clinical value of detecting granins in tissues and measuring circulating levels of granins, particularly chromogranin A. In addition to providing information about the neuroendocrine character of various neoplasms, measurement of chromogranin A has yielded insights into the pathogenesis of essential hypertension.

molecular and genetic aspects of granins

structural and physicochemical properties Granins consist of single-polypeptide chains of approximately 180 to 700 amino acid residues, bearing an amino-terminal signal peptide that directs the movement of the preproteins from ribosomes to the endoplasmic reticular lumen and, hence, the Golgi complex, where further post-translational modifications occur. Granins tend to bind calcium with low affinity but high capacity and then aggregate in vitro at low pH in the presence of calcium.18-24 These aggregation characteristics suggest that granins have functions within the core of secretory granules.

genomic organization and transcriptional regulation The chromosomal positions of all granins except secretogranin IV have been determined in humans, cows, mice, and rats (Table 1); in each case, the loci lie in regions of locally

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mechanisms of disease conserved gene order in homologous chromosomes (Fig. 2).25 A pH The distribution of granins throughout the neu- 5.8 5.4 5.0 4.6 4.2 roendocrine system has been studied with Northern blot analysis of messenger RNA. Granin messages CgB have been found in cells of various neuroendocrine DBH CgA tissues that have a regulated secretory pathway.9,26,27 97 Moreover, granin synthesis responds differently in different cell types to agents that elevate cyclic 66 AMP (cAMP), steroid hormones, neurotrophins, and phorbol ester. Experiments with transfected granin-gene pro- 45 moters have clarified the mechanism of constitutive and secretagogue-inducible expression of granins in specific cells.28 The proximal promoter regions of chromogranin A, chromogranin B, and secreto- 24 granin II contain a functional cAMP-response ele- Molecular Mass (kD) ment (CRE) upstream of a TATA box (a region of seven nucleotides, mainly thymidine and adenine, located upstream of the starting point of transcrip- 18 tion); otherwise, their promoter sequences dif- fer.29,30 The specificity of expression of chromogranin A, chromogranin B, and secretogranin III in mouse neuroendocrine cells has been mapped to the CRE site.30-32 Upstream promoter elements may also be important in cell-specific expression of hu- B C man chromogranin A and secretogranin II.33,34 These CRE sites may explain how the expression of chromogranin A, chromogranin B, and secretogranin II is up-regulated in neuroendocrine cells by nicotinic–cholinergic agonists and the pregangli- onic neuropeptide pituitary adenylyl cyclase–activating polypeptide.31,32,35,36 Moreover, neuronal differentiation of a pheochromocytoma cell line 100 nm 5 mm (PC12) induced by neurotrophin nerve growth factor up-regulates expression of the genes for chromogranin A and chromogranin B through an effect Figure 1. Localization of Chromogranins with Dense-Core Secretory Granules on the CRE site.31,37 of Sympathoadrenal Chromaffin Cells. An upstream serum response element also has Panel A shows soluble-core proteins in bovine chromaffin vesicles after two- an important role in the expression of the mouse dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis, fol- 32 lowed by Coomassie blue staining. DBH denotes dopamine b-hydroxylase, CgA secretogranin II gene, and a proximal G- or chromogranin A, and CgB chromogranin B. Panel B shows an electron micro- C-rich region contributes to the expression of the graph of bovine chromaffin cells. The electron-dense spherical and oblong struc- mouse chromogranin B gene.31 Glucocorticoid tures are dense-core secretory chromaffin granules. The black particles, which have a diameter of 8 to 12 nm, represent immunogold labeling of chromogranin sensitivity has been mapped to a novel glucocorti- A with rabbit antibovine chromogranin A antibody. Panel C shows the subcellular coid-response-element variant in the rat chromo- distribution of a human chromogranin A–enhanced green fluorescent protein granin A.38 (EGFP) chimeric photoprotein, in living sympathoadrenal PC12 cells. Chromo- granin A–EGFP expression was examined by three-dimensional deconvolution microscopy. Nuclei were visualized with blue dye (Hoechst 33342). Optical sec- sorting mechanisms tions along the z axis were acquired with increments of 200 nm and the use of Proteins are secreted from cells by exocytosis in ei- a 100¬ oil-immersion objective to generate three-dimensional views of the ther a constitutive or a regulated manner.39,40 The photoprotein distribution. Chromogranin A–EGFP displays a bright, punctate or vesicular fluorescence signal, especially within the subplasmalemmal re- rate of secretion through the constitutive pathway, gion, indicating storage of the chimera in chromaffin secretory granules. which operates in every type of cell, is a simple func-

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Table 1. Physicochemical Properties of Granins.*

SgIII SgIV SgV SgVI Property CgA CgB SgII (1B1075) (HISL-19) (7B2) (NESP55)

Chromosome localization 14 (human), 20 (human), 2 (human), 2 (mouse) ND 15 (human), 20 (human) 21 (bovine), 3 (rat), 9 (rat), 2 (mouse) 6 (rat), 2 (mouse) 1 (mouse) 12 (mouse) Amino acid residues† 431–445 626–657 559–586 449–507 ND 185 241 Molecular mass (kD)‡ Calculated 49–52 48–52 67.5 51–57 ND 21 27.5 Apparent 74–80 100–120 86 57 35 23 55 Acidic residues (%) 25 24 20 19 ND 16 21 Isoelectric point (pHi) 4.5–5.0 5.1–5.2 5.0 5.1 5.6 5.2 4.4–5.2 Multibasic sites§ 8–10 15–18 9 6–10 ND 3 5 Disulfide-bonded loop Yes Yes No No ND No No Calcium binding Yes Yes Yes ND ND Yes ND Thermostability Yes Yes Yes ND ND Yes Yes Phosphorylation Yes Yes Yes ND ND Yes Yes Sulfation Yes Yes Yes Yes ND Yes ND O-glycosylation Yes Yes Yes ND ND ND Yes N-glycosylation No Yes No No No No No Phosphorylation Yes Yes Yes ND ND Yes Yes

* CgA denotes chromogranin A, CgB chromogranin B, SgII secretogranin II, SgIII secretogranin III, SgIV secretogranin IV, SgV secretogranin V, SgVI secretogranin VI, and ND not determined. † Amino acid residues are for mature protein without signal peptide. ‡ The molecular mass was calculated from the primary structure. The apparent molecular mass was determined with the use of sodium dodecyl sulfate–polyacrylamide-gel electrophoresis. § Multibasic sites refers to sites with two or more consecutive Arg or Lys residues.

tion of the rate of synthesis of the secreted sub- nonretained proteins.41 The mechanism by which stance. In this pathway, newly synthesized proteins granins are sorted at the trans-Golgi network to en- (e.g., albumin and immunoglobulins) continuous- ter the regulated pathway of secretion is unclear.41-48 ly pass through to the trans-Golgi network and are Selective aggregation of regulated secretory then transported in constitutive vesicles to the plas- proteins at the level of the trans-Golgi network ma membrane for immediate release. In contrast, (“sorting by retention”) may occur under conditions the regulated secretory pathway operates in special- of high Ca2+ and acidic pH. This mechanism seg- ized cells, such as neuroendocrine cells and neurons. regates the regulated cargo from constitutively se- In this pathway, secretory vesicles (frequently called creted proteins and prevents the escape of regulat- “secretory granules”) containing a condensed cargo ed secretory proteins from immature granules into of peptide or amine hormones or neurotransmit- the constitutive-like secretory pathway.41 For chro- ters may remain in the cell for extended periods of mogranin A, chromogranin B, and secretogranin II, time. These granules release their contents only in this aggregation depends on millimolar calcium response to a stimulus that is specific for a particu- concentrations and a mildly acidic pH, conditions lar type of cell (e.g., acetylcholine for the chromaf- that are fulfilled in the lumen of the trans-Golgi fin cell). There is a third, constitutive-like secretory network.21,22,43 pathway for protein sorting within immature secre- A specific sorting signal in the secretory protein tory granules, leading to constitutive secretion of that binds to a sorting receptor may facilitate the

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mechanisms of disease

Chromogranin A I II III IV V VI VII VIII

Chromogranin B I II III IV V

Secretogranin II I II

Untranslated domain

Signal-peptide domain

Translated domain Disulfide-bonded loop domain

Homologous C-terminal domain

Figure 2. Comparison of the Organization of the Genes Encoding Chromogranin A, Chromogranin B, and Secretogranin II and Homologous Domains within Each Protein. Roman numerals designate exon numbers. The genes are not drawn to scale. movement of the protein to the regulated pathway A appears to be crucial for the formation of secreto- of secretion (“sorting for entry”).41,44,45 Indeed, ry granules and sequestration of hormones in neu- chromogranin B contains a hydrophobic loop struc- roendocrine cells.58 Impairment of chromogranin ture, which may guide the sorting of chromogranin A expression by antisense RNA depletes secretory B from the trans-Golgi network to secretory gran- granules, inhibits regulated secretion of a pro- ules of neuroendocrine PC12 pheochromocytoma hormone, and reduces secretory granule protein in cells.49-52 Chromogranin A also contains such a loop cells. Reestablishment of the regulated secretory structure. phenotype in chromogranin A–deficient cells has been achieved with the reintroduction of chromo- 58 intracellular functions granin A by transfection. of granins modulation of peptide hormone formation of secretory granules and neuropeptide processing Several granins undergo aggregation induced by Granins are proproteins, with multiple recognition low pH and high calcium levels and interact with sites for endopeptidases, such as prohormone con- other components of the matrix of the secretory vertase 1 and 2 (PC1 and PC2), which are serine en- granule, such as catecholamines, serotonin, and his- doproteases, and plasmin.59-62 A neuroendocrine tamine, suggesting that granins contribute to the peptide from the carboxy-terminal of secretogranin formation of secretory granules.21-24,53-55 The pre- V (7B2) selectively inhibits the prohormone conver- vailing view is that granins contribute to the forma- tase PC2, whereas the amino-terminal domain of tion of the secretory vesicle when the immature ves- 7B2 acts as a PC2 chaperone in the endoplasmic icle buds from the trans-Golgi network. Granins reticulum, where it may be required for pro-PC2 ac- facilitate the formation of storage complexes and tivation.13 In 7B2-knockout mice, which have no function as chaperones in the sorting of other reg- demonstrable PC2 activity, the processing of pan- ulated secretory proteins.21,22,55-57 Chromogranin creatic-islet hormones is deficient, and such mice

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have hypoglycemia, hyperproinsulinemia, and hy- cells.65,66 In humans, exogenous pancreastatin di- poglucagonemia.63 minishes glucose uptake by skeletal muscle.67 Pan- creastatin also activates hepatic glycogenolysis and extracellular functions inhibits insulin signaling in adipocytes — effects that seem to depend on activation of a phospholip- autocrine and paracrine inhibition ase by particular subunit isoforms of heterotri- of secretion meric G proteins65,66; however, a unique G protein– The presence of numerous paired basic amino ac- coupled receptor for pancreastatin has not been ids in granins suggests that they function as prohor- identified. In addition, pancreastatin inhibits the mones, giving rise to bioactive peptides as a result release of amylase from exocrine pancreas, the re- of post-translational proteolytic processing. Indeed, lease of gastric acid from parietal cells, and the re- peptides derived from chromogranin A, chromo- lease of parathyroid hormone from parathyroid granin B, and secretogranin II have autocrine, para- chief cells.65,66 crine, and endocrine activities (Fig. 3 and Table 2). Chromogranin A is also the precursor of amino- One such peptide is chromogranin A–derived pan- terminal fragments (vasostatins I and II), which creastatin from porcine pancreas.64 Pancreastatin inhibit vasoconstriction in isolated human blood elevates blood glucose by inhibiting glucose-stim- vessels68 and modulate the adhesion of fibroblasts ulated insulin release from pancreatic islet beta and coronary-artery smooth muscle cells.69 Sup-

Primary transcript I II III IV V VI VII VIII Exon

I II III IV V VI VII VIII Mature mRNA ¡18 ¡4 1344 67 100 251 412 439

Chromogranin A ss 100 200 300 400 Functional peptides CgA 1–40Chromacin 176–197 Catestatin 352–372

s s Vasostatin I (1–76) Prochromacin 79–439

s s

Pancreastatin Parastatin Vasostatin II (1–115) 250–301 357–428

s s

Untranslated region Paired basic residues Signal-peptide domain Oligoglutamate region

Disulfide-bonded loop domain Intron s s

Figure 3. Peptide-Encoding Regions and Putative Functional Domains of Human Chromogranin A (CgA). Arabic numbers designate amino acids in the mature protein (minus signal peptide). Roman numerals designate exon numbers. The intron– exon structure is not drawn to scale.

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Table 2. Proposed Actions of Granin-Derived Peptides.

Fragment and Position* Biologic Activity of the Fragment or Its Orthologue

Chromogranin A Bovine CgA 1–40 Increases release of calcitonin and calcitonin gene–related peptide from lung tumor cells, inhibits vasoconstriction, inhibits parathyroid hormone secretion from parathyroid chief cells Chromofungin (bovine CgA 47–66) Exerts antifungal activity Vasostatin I (bovine CgA 1–76) Inhibits vasoconstriction, promotes fibroblast adhesion, inhibits parathyroid hormone secretion from parathyroid chief cells, triggers microglial-cell–mediated neuronal apoptosis and exerts bacteriolytic and antifungal effects Vasostatin II (bovine CgA 1–113) Inhibits vasoconstriction and parathyroid hormone secretion Prochromacin (bovine CgA 79–431) Exerts bacteriolytic and antifungal effects Chromacin I and II (bovine CgA 173–194 Exerts bacteriolytic and antifungal effects and bovine CgA 195–221) Pancreastatin (porcine CgA 240–288) Inhibits insulin release from pancreatic-islet beta cells; promotes hepatic glycogenolysis; decreases insulin-induced glycogen synthesis in skeletal myocytes, hepatocytes, and adipocytes; stimulates amylase release from pancreatic acini; decreases gastric acid release from parietal cells; diminishes glucose uptake by skeletal muscles in humans Catestatin (bovine CgA 344–364) Inhibits catecholamine release from the adrenal medulla Parastatin (porcine CgA 347–419) Inhibits parathyroid hormone secretion from parathyroid chief cells Chromogranin B Bovine CgB 1–41 Inhibits parathyroid hormone secretion from parathyroid chief cells Chrombacin (bovine CgB 564–626) Exerts bacteriolytic effects Secretolytin (bovine CgB 614–626) Exerts bacteriolytic effects Mouse CgB 1–657 Inhibits biosynthesis and release of insulin Secretogranin II Secretoneurin (rat SgII 154–186) Stimulates dopamine release from central striatal neurons and basal ganglia; stimulates gonadotropin II secretion from pituitary; inhibits serotonin and melatonin release from pinealo- cytes; promotes chemotactic attraction of monocytes, eosinophils, and fibroblasts; stimulates proliferation and migration of vascular smooth-muscle cells; stimulates migration and inhibits proliferation of endothelial cells; stimulates transendothelial migration of monocytes; activates endothelial cells for neutrophil adherence Secretogranin V Amino-terminal (NT) peptide (human 7B2 1–135) Acts as activator and chaperone of pre-prohormone convertase 2 Carboxy-terminal (CT) peptide (human 7B2 Inhibits prohormone convertase 2 155–185) Secretogranin VI Bovine SgVI 159–162 Acts as antagonist of 5-HT1B serotonergic receptor

* The actions of secretogranin III and IV have not been described. CgA denotes chromogranin A, CgB chromogranin B, SgII secretogranin II, and SgVI secretogranin VI.

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pression of the release of parathyroid hormone ments induce a neurotoxic phenotype in brain mac- has been reported in association with the chromo- rophages (microglia), which triggers apoptotic de- granin A fragments vasostatin70 and parastatin.71 generation of cortical neurons.85-87 A secretogranin Chromogranin A has also been reported to inhibit II–derived peptide, secretoneurin, stimulates the re- secretion of proopiomelanocortin hormone,72 but lease of dopamine from nigrostriatal neurons and such activity has yet to be ascribed to a discrete re- has chemoattractant effects on monocytes, eosin- gion of the protein. ophils, fibroblasts, vascular smooth-muscle cells, Catestatin, another fragment of chromogranin and endothelial cells.9,88 A, inhibits the release of catecholamines from sympathoadrenal chromaffin cells by blocking the neu- distribution in normal ronal nicotinic cholinergic receptor, which is the neuroendocrine tissues physiologic trigger for secretion (Fig. 4).73-76 Cate- statin also prevents the desensitization of cate- The widespread distribution of granins within the cholamine release from chromaffin cells that is in- endocrine, neuroendocrine, and central and periph- duced by repeated nicotinic-agonist stimulation.76 eral nervous systems is now firmly established. In Thus, catestatin may contribute to an autocrine the central nervous system, chromogranin A is de- negative-feedback mechanism that modulates cat- tectable in neurons in the cerebellum, cerebral cor- echolamine release within the sympathoadrenal tex, septum, and amygdala and perhaps in astroglial system. Since excess sympathetic activity has been cells.3,89-91 Chromogranin B and secretogranin II implicated in the development of hypertension, a are similarly distributed, though their quantity var- disturbance of the catestatin mechanism may be a ies according to the type of neuroendocrine cell.9,27 contributing factor. Indeed, recent studies show There is also widespread distribution of secreto- that the plasma catestatin level is diminished in pa- granin V in the central nervous system and various tients with hypertension and even in normotensive neuroendocrine tissues13; secretogranin VI is present persons at genetic risk for hypertension. The obser- in the central nervous system, adrenal medulla, an- vation that a low catestatin level is correlated with terior and posterior pituitary, and intestine.14 increased adrenal epinephrine secretion and augmented pressor responses to sympathoadrenal clinical uses stressors provides further evidence of a link between diminished catestatin and hypertension.77 Numerous studies have established that granins Some of the responses of secretoneurin and pan- can be detected in an array of endocrine, neuroendo- creastatin are impaired by pertussis toxin, suggest- crine, and neuronal tumors (Table 3), from which ing a role of G protein–coupled receptors.9,65,66,78 they are secreted into the bloodstream.92 The distri- However, unique receptors have not been identified bution of granins in neoplasms is generally corre- for most granin fragments.66,78,79 Catestatin seems lated with their expression in the corresponding to act by binding directly to the nicotinic cholinergic normal tissue. receptor,74-76 whereas vasostatin may relax vascular smooth-muscle myocytes by opening hyperpolar- assays for chromogranin a izing cell-surface potassium channels.79 A competitive radioimmunoassay can detect circulating chromogranin A, with the use of purified full- antimicrobial properties of granins length human chromogranin A.92,93 Plasma chro- During stress, granins are released through exocy- mogranin A immunoreactivity is remarkably stable tosis into the blood from both the sympathoadre- in vitro, readily surviving prolonged heating at nal system and the anterior pituitary gland.80,81 37°C, as well as repeated freezing and thawing.93 These granins and their fragments may have a role Several other radioimmunoassays94,95 and enzyme in systemic infection. The antibacterial and anti- immunoassays96,97 have been developed for the fungal activities of fragments of chromogranin A measurement of granins or granin-derived peptides (prochromacin, chromacin I, and chromacin II) re- in plasma or serum.16,77,94,98 In general, the meas- sult from the ability of these fragments to form ion urement of intact chromogranin A in plasma has channels through membranes.82,83 The chromo- greater sensitivity for the diagnosis of neuroendo- granin B fragment secretolytin also has bacteriolytic crine tumors than the measurement of fragments.16 properties.84 Chromogranin A or its vasostatin frag- The results of enzyme immunoassays and radioim-

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Nicotinic cholinergic Extracellular agonist binding site + Na+ – Ca2+ Voltage- Catestatin gated Nicotinic calcium cholinergic Catecholamines channel receptor Chromogranins, secretogranins Neuropeptides

Cell membrane +

Membrane Exocytosis depolarization Cytoplasm Ca2+ Na+ Fusion

Docking

Golgi complex Chromogranin A processing

Transcription Chromaffin Translation granule Sorting

Nucleus Chromaffin cell

Chromogranin A gene

Figure 4. Autocrine–Paracrine Regulation of Catecholamine Release from Sympathoadrenal Chromaffin Cells by Catestatin. The physiologic secretagogue acetylcholine binds to the agonist pocket on the nicotinic cholinergic receptor, triggering sodium influx and consequent membrane depolarization, which activates calcium influx through voltage-gated channels and leads to exocytotic release of the chromaffin-granule cargo. After the cargo has been released, catestatin exerts po- tent antagonistic effects on nicotinic cholinergic signaling, resulting in negative-feedback modulation of catecholamine release. Arrows with plus signs indicate stimulation, and the arrow with a minus sign inhibition. munoassays are closely correlated.94 The limit of three radioimmunoassay kits for the detection of detection may be lower with enzyme immunoas- chromogranin A are available commercially.16,94,95 says than with radioimmunoassays, but radioim- Internet-based protocols for human chromogranin munoassays cover a wider range of concentrations.94 A radioimmunoassay are available free of cost In addition to the availability of granin assays from for research purposes (http://medicine.ucsd.edu/ reference laboratories for clinical samples,93,99 hypertension).

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Table 3. Detection of Granins in Human Neuroendocrine Tumors.*

SgIII SgIV SgV Site and Type of Tumor CgA CgB SgII (1B1075) (HISL-19) (7B2)

Adrenal medulla Pheochromocytoma + + + ND ND + Anterior pituitary Corticotropinoma + + + ND ND + Gonadotropinoma + + + ND ND ND Somatotropinoma + + + ND ND + Thyrotropinoma + + + ND ND ND Prolactinoma ¡ + + ND ND + Nonfunctioning adenoma + + + ND ND + Carcinoid Liver + + ND ND ND ND Lung + + + ND + + Middle ear + ND ND ND ND ND Ovary + ND ND ND ND ND Prostate + + + ND ND ND Thymus + ND ND ND ND ND Uterus + ND ND ND ND ND Gastroenteropancreatic Carcinoid + + + ND ND ND Gastrinoma + + ND ND ND ND Glucagonoma + + + ND + + Insulinoma + + + ND + + PPoma + ND ND ND + + Somatostatinoma + ND ND ND ND ND VIPoma + ND ND ND ND ND Nonfunctioning islet-cell carcinoma + ND ND ND ND + Cardiovascular Aortic body + ND ND ND ND ND Carotid body + ND ND ND + ND Parathyroid Adenoma + + + ND ND ND Carcinoma + ND ND ND ND ND Hyperplasia + + ND ND ND ND

use of granins as diagnostic markers agnostic of these neoplasms.3,15 Measurement of or biologic markers chromogranin A levels in blood can also be used to Chromogranin A is a standard probe for immuno- monitor the progression or regression of neuroen- histochemical analyses of neuroendocrine tumors, docrine tumors during treatment.99-101 and elevated serum chromogranin A levels are di- Measurement of plasma chromogranin A in

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Table 3. (Continued.)

SgIII SgIV SgV Site and Type of Tumor CgA CgB SgII (1B1075) (HISL-19) (7B2)

Neural Ganglioneuroblastoma + ND ND ND ND ND Ganglioneuroma + ND + ND ND ND Neuroblastoma + + + ND ND ND Medulloblastoma + ND ND ND ND ND Paraganglioma + + + ND ND ND Skin Merkel-cell tumor + ¡ + ND + ND Medullary thyroid Carcinoma + + + ND + + Hyperplasia + + + ND + ND Tumors with variable neuroendocrine differentiation Prostate + + + + ND ND Breast + + + ND ND ND

* A plus sign denotes detectable immunoreactivity, the presence of messenger RNA for the granin, or both; a minus sign denotes the absence of either detectable immunoreactivity or messenger RNA. ND denotes not determined. No data are available for secretogranin VI (NESP55) immunoreactivity in human tumors. CgA denotes chromogranin A, CgB chromogranin B, SgII secretogranin II, SgIII secretogranin III, SgIV secretogranin IV, SgV secretogranin V, PP pancreatic polypeptide, and VIP vasoactive intestinal polypeptide. children with suspected neuroblastoma has a sen- of the chromogranin B–derived peptides GAWK sitivity of 91 percent and a specificity of 100 percent and CCB are elevated in patients with pancreatic for the diagnosis,102 and since chromogranin A islet-cell tumors105,106 and those with bronchial levels are correlated with the tumor burden (or tumors.107 Plasma secretoneurin (a secretogranin II stage of disease), the test can be used to monitor fragment) may be elevated in patients with gastro- the response to treatment and to predict survival. enteropancreatic neuroendocrine tumors or pheo- Chromogranin A levels may also be elevated in pa- chromocytomas.98 Elevated plasma secretogranin tients with primary parathyroid hyperplasia,15 thy- V levels are associated with several types of neuro- roid C-cell hyperplasia,15 or gastric enterochromaf- endocrine tumors13,108-111 and small-cell lung fin-like cell hyperplasia.103,104 For this reason, an carcinoma.109,112 increased chromogranin A level may not reliably distinguish neuroendocrine hyperplasia from ade- pheochromocytoma noma or carcinoma. Several circulating granins are sensitive biologic Immunohistochemical detection and plasma markers for pheochromocytoma. An elevated plas- quantification of other granins and granin-derived ma chromogranin A level may be diagnostic of phe- peptides may be of diagnostic importance. Immuno- ochromocytoma, von Hippel–Lindau disease,113,114 reactivity for chromogranin B and, to some extent, multiple endocrine neoplasia type II, or neurofibro- secretogranin II, III, IV, and V has been documented matosis. In sporadic cases of pheochromocytoma, in various neuroendocrine tumors (Table 3), and cir- elevation of the plasma chromogranin A level has a culating levels of these granins may reflect the secre- sensitivity of 83 percent and a specificity of 96 per- tory activity of the tumor. Prolactin-producing tumor cent for the diagnosis, and the chromogranin A cells of the anterior pituitary are negative for chro- level is correlated with the tumor mass.115 Drugs mogranin A but are positive for both chromogran- commonly used to diagnose or treat pheochromo- in B and secretogranin II (Table 3), and plasma levels cytoma, such as phentolamine, tyramine, clonidine,

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and metoprolol, do not substantially alter the serum for monitoring such patients.122 In patients with chromogranin A level. Furthermore, unlike plasma gastrinoma (the Zollinger–Ellison syndrome), the epinephrine and norepinephrine levels, the plasma role of chromogranin A is not well defined. Elevated chromogranin A level can be used to distinguish levels of chromogranin A appear to reflect the asso- between malignant and benign pheochromocyto- ciated gastrin-mediated enterochromaffin-like cell mas. In one study, patients with benign disease had hyperplasia,123 rather than the actual size of the a mean (±SE) chromogranin A level of 188±40.6 ng gastrinoma, and excision of the stomach alone sub- per milliliter, whereas those with malignant disease stantially reduces the plasma chromogranin A lev- had a mean chromogranin A level of 2932±900 ng el, even without excision of the gastrinoma.103 Al- per milliliter.100 Elevated levels of serum chromo- though the chromogranin A level may be elevated granin A may not be specific for the diagnosis of in patients with primary hyperparathyroidism, it pheochromocytoma in patients with impaired re- is most likely to be elevated in those who also have nal function.93,116 However, this limitation can be the Zollinger–Ellison syndrome, and in such pa- overcome by evaluating renal function93,116,117 and tients, parathyroidectomy is especially likely to low- combining the measurement of chromogranin A er the plasma chromogranin A level.124 with that of plasma catecholamines, which may have a lower diagnostic sensitivity but have a high- lung, prostate, colon, and breast tumors er specificity and positive predictive value.117 A nor- In patients who have small-cell lung cancers with mal plasma chromogranin A level may be a valuable neuroendocrine differentiation, the plasma chro- clue to the diagnosis of factitious pheochromocy- mogranin A level may serve as a marker of the re- toma.118 Measurements of chromogranin B and sponse to treatment and may be useful in moni- secretogranin II may also be of diagnostic value in toring patients for recurrent disease. Although the patients with suspected pheochromocytoma. In one chromogranin A level indicates the likely degree study, plasma chromogranin B levels were elevated of neuroendocrine differentiation within lung tu- in 81 percent of such patients,119 and another study mors, its low sensitivity makes it unreliable as a di- showed that the plasma secretoneurin level was in- agnostic marker of such neoplasms.15,122,125 creased by a factor of 4 to 5.98 In men with prostate cancer, measurement of the chromogranin A level may be useful in estab- carcinoid tumors lishing the diagnosis and determining the progno- The highest levels of serum chromogranin A (up to sis.126,127 Circulating chromogranin A levels may 1000 times the upper limit of the normal range) be increased even if the prostate-specific antigen have been found in patients with metastatic carci- level is normal.128 An elevated chromogranin A lev- noid tumors.15 The stability of serum chromogran- el, possibly because it is a marker of neuroendocrine in A speaks favorably for its use in detecting carci- differentiation, may also predict a lack of response noid tumors and monitoring their progression; of the tumor to hormone therapy; indeed, elevated alternative diagnostic tests for carcinoid tumors levels may portend a poor prognosis.129 include tests of urinary 5-hydroxyindoleacetic acid, Chromogranin A immunoreactivity has been serum serotonin (5-hydroxytryptamine), and serum reported in some cells in colorectal tumors130 and neuron-specific enolase. In multiple endocrine neo- breast tumors,131 but its diagnostic or prognostic plasia type I, there is a clear correlation between the value has not been established. Several other tumors tumor mass and the circulating level of chromo- with suspected or partial neuroendocrine differen- granin A.120 In patients with midgut carcinoid tu- tiation or lineage (choriocarcinoma, thymoma, ma- mors, an elevated chromogranin A level is an inde- lignant melanoma, and renal-cell carcinoma) are pendent predictor of death.101,121 not associated with an elevated plasma chromogranin A level.93,121 endocrine pancreatic tumors Two thirds of patients with endocrine pancreatic nonfunctioning neuroendocrine tumors tumors have clinically “functional” tumors that se- Chromogranin A can also aid in the diagnosis of crete hormonal markers. In patients with tumors clinically silent, or “nonfunctioning,” neuroendo- that secrete glucagon, somatostatin, or vasoactive crine tumors.17,132-134 Indeed, cases of medullary intestinal polypeptide, serum chromogranin A lev- thyroid carcinoma, anterior pituitary adenoma, els are usually elevated and serve as tumor markers small-cell lung cancer, and islet-cell carcinoma that

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Downloaded from www.nejm.org by RODRIGUEZ POVEDA AIXA MILDRED on May 16, 2010 . Copyright © 2003 Massachusetts Medical Society. All rights reserved. mechanisms of disease are hormone-negative but chromogranin A–posi- and in the swollen neurons of Pick’s disease.140,141 tive have been reported.133,134 Studies suggest that chromogranin A activates brain microglial cells, perhaps contributing to neuronal essential hypertension degeneration.85-87 Increased sympathoadrenal activity may have a Cerebrospinal fluid chromogranin A and chro- causative role in essential (idiopathic, familial, or mogranin B levels are reduced in patients with genetic) hypertension. The release of chromogranin schizophrenia.142 Genetic linkage studies of schizo- A with catecholamines indicates that exocytosis is phrenia in Japan implicate a genomic region near the mechanism of physiologic catecholamine re- the chromogranin B locus on chromosome 20 lease in humans.80 The basal plasma chromogranin (CHGB),143 and allelic-association studies in Chi- A level is correlated with sympathetic tone,135,136 na have associated single-nucleotide polymorphisms and studies in twins indicate that the basal level is at the CHGB locus with schizophrenia.144 highly heritable.137 As compared with age-matched normotensive controls, patients with essential hy- organ failure pertension have an increased plasma chromogranin Chromogranin A levels are elevated in patients A level and an increased release of stored chromo- with kidney, liver, or heart failure. As a result of the granin A in response to insulin-evoked hypoglyce- retention of midmolecule fragments, chromogran- mia.137 The dysglycemic chromogranin A fragment in A levels in patients with end-stage renal disease pancreastatin is also elevated in patients with es- can be as high as those in patients with neuroendo- sential hypertension138; its actions may therefore crine neoplasia, suggesting that the kidneys have contribute to the insulin resistance that often ac- an important role in eliminating chromogranin companies the condition. Catestatin, the chromo- A.116 The moderate elevation of chromogranin A granin A–derived peptide that inhibits catechola- levels in patients with hepatic failure suggests that mine release, is decreased in patients with essential either the liver metabolizes chromogranin A93 or hypertension and even in normotensive subjects the neuroendocrine system is activated in such pa- with a family history of hypertension.77 Moreover, tients. Neuroendocrine activation also occurs in people with such a family history have increased patients with congestive heart failure, and the plas- epinephrine secretion in addition to diminished ma chromogranin A level rises as a result of this catestatin, suggesting an inhibitory effect of cate- activation. Moreover, the chromogranin A level is statin on chromaffin cells in vivo.77 Low catestatin directly correlated with the severity of the disease. levels also predict augmented adrenergic responses In patients with New York Heart Association class to stressors, suggesting that a reduction in cate- IV heart failure, the chromogranin A level is approx- statin increases the risk of hypertension. imately 7.6 times the normal level.145 cerebrospinal fluid levels conclusions and neuropsychiatric diseases Chromogranin A is present in the cerebrospinal The study of granins was initiated over 30 years ago, fluid and appears to derive from the central nerv- after the discovery of the prototype, chromogranin ous system rather than a peripheral source. Where- A,5,6 and in the past 15 years, our knowledge of the as plasma chromogranin A responds to alterations structure and function of granins has increased dra- of peripheral sympathoadrenal activity, cerebro- matically. There is compelling new evidence of the spinal fluid chromogranin A is not correlated with intracellular and extracellular functions of these central noradrenergic neuronal activity.139 In pa- proteins. Within the cells of origin, a granulogenic tients with Parkinson’s disease, the cerebrospinal or sorting role in the regulated pathway of hormone fluid chromogranin A level is diminished, which or neurotransmitter secretion has been document- may be a useful diagnostic finding; however, the ed, especially in the case of chromogranin A. Granins chromogranin A level does not increase after adre- also function as prohormones, giving rise by prote- nal-to-caudate autografting of chromaffin cells in olytic processing to a large array of peptide frag- such patients.139 ments for which diverse autocrine, paracrine, and Chromogranin A accumulates in the senile and endocrine activities have been demonstrated. How- preamyloid plaques of Alzheimer’s disease, in Lewy ever, more definitive insights into granin functions bodies in the substantia nigra in Parkinson’s disease, are required, and they may be achieved in the near

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future with the use of strategies such as single or renal overactivity, which in turn leads to the devel- multiple granin-gene deletions or knockouts by tar- opment of the syndrome. geted homologous recombination. Supported by grants from the National Institutes of Health The emerging technique of RNA-mediated in- (DK59628, to Dr. Taupenot, and HL58120 and HL69758, to Dr. O’Connor) and the Department of Veterans Affairs (to Dr. O’Connor). terference with gene expression in mammalian cells We are indebted to Drs. Peter E. Cadman, Bruce M. Gill, Vivian Y. and living animals may be of value for determining Hook, Qijiao Jiang, Mala T. Kailasam, Brian P. Kennedy, Nitish R. the loss-of-function phenotypes for the granins. Mahapatra, Manjula Mahata, Sushil K. Mahata, Robert J. Parmer, Fangwen Rao, David J. Rozansky, Mats Stridsberg, Marwan A. Tak- Measurements of chromogranin A fragments in pa- iyyuddin, Kechun Tang, Carolyn V. Livsey-Taylor, Hongjiang Wu, Suk- tients with essential hypertension suggest that a de- kid Yasothornsrikul, Noboru Yanaihara, and Michael G. Ziegler for ficiency of catestatin, a fragment that inhibits the their collaboration, and to Dr. Åsa Thureson-Klein for the electron micrograph of chromogranin A in chromaffin granules of the adre- release of catecholamines, triggers sympathoad- nal medulla.

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