Daniele Tota.
Chromogranin A: Functions and clinical applications.
Granin is a protein family that are stored and released in vesicles throughout the neuroendocrine system and in a variety of neurons. Granins include Chromogranin and Secretogranins a unique group of acidic, soluble secretory proteins. Chromogranin A (CgA) is a member of the chromogranin family and it is the first to be discovered and the well characterized.
CgA is one of the most important biomarkers for the diagnosis of Neuroendocrine tumors (NETs), a form of cancer that differs from other neoplasia in that they synthesize, store, and secrete peptides and amines.
THE GRANIN FAMILY.
Granins consist of single-polypeptide chains of approximately 180 to 700 amino acid residues, with an amino-terminal signal peptides that directs the movement of the preproteins from ribosomes to endoplasmatic reticular lumen and hence, the Golgi complex, where post-translational modifications occur. Granins tend to bind calcium with low affinity but high capacity and it suggests that granins have functions within the core of secretory granules. Granins are major components of large dense-core secretory vesicles.
The granin family comprises eight members: CgA, CgB, CgC (Secretogranin II [SgII]), SgIII, SgIV, SgV (7BS), SgVI (NESP55) and VGF.
Chromogranin A—Biological Function and Clinical Utility in Neuro Endocrine Tumor Disease, 2010.
CgA. It was the first member of granin family to be identified and it is expressed in all type of neurons. CgA is heat stable, hydrophilic and acid protein, composed by 460 amino acids with a molecular mass of 70 to 85 kDa. Its N-terminal region contains a disulfure bridged formed by two cysteine residues.
CgA biosynthesis is controlled both transcriptionally and post-transcriptionally.
Post-translational processing results in a series of smaller biological active peptides such as pancreastatin (corresponding to CgA residues 250–301), catestatin (corresponding to CgA residues 352–372), and vasostatin I and II (corresponding to CgA residues 1–76 and 1–113, respectively).
CgB and CgC. Both are considered to play roles in regulating secretion.
CgB is a major granin component in adrenal medulla and may be a more sensitive marker for pheochromocytoma. In addition, CgB, unlike CgA, can localize to the nucleus and has been reported to modulate gene expression. CgB was shown to increase the transcription of zinc finger protein, while suppressing that of T3 -receptor, troponin C, and integrin.
CgC has a unique role action that includes effects on genes involved in notch signaling and the guanylate cyclase pathway.
Its principle prohormone processing product, secretoneurin, is found in its fully processed and bioactive form in blood as a large fraction of total SgII plasma immunoreactivity.
The Chromogranin–Secretogranin Family, 2003.
Is secretoneurin a new hormone?, 2012.
Others secretogranine. About SIII no biologically active peptides derived from SgIII have been described. 7BS functions as a chaperone of Prohormone convertase 2 (PC2) is the main processing enzyme in pancreatic alpha cells. NESP55 is part of the extremely complex imprinted GNAS gene locus which encodes the α-subunit of the stimulatory G protein. Post-translational processing of a single VGF precursor gives raise to a varied multiplicity of neuro-endocrine peptides, some of which are secreted upon stimulation both in vitro and in vivo. Several VGF peptides, accounting for ∼20% of the VGF precursor sequence, have shown biological roles including regulation of food intake, energy balance, reproductive and homeostatic mechanisms, synaptic strengthening, long-term potentiation (LTP) and anti-depressant activity.
VGF: an inducible gene product, precursor of a diverse array of neuro-endocrine peptides and tissue-specific disease biomarkers, 2011.
Intracellular functions of granins.
The constitutive pathway operates in every type of cells and is a function of the rate of synthesis of the secreted substance; newly synthesized proteins pass through to the trans-Golgi network and are transported in constitutive vesicles to the plasmatic membrane for immediate release. In contrast, the regulated secretory pathway operates in specialized cells, as neuroendocrine cells and neurons; the secretory vesicles containing a condensed cargo of peptide or amine hormone or neurotransmitters may remain in the cell for extended periods of time before to be secreted in response to a specific stimulus. Granins contribute to the formation of secretory granules. Chromogranin A appears to be crucial for the formation of secretory granules and sequestration of hormones in neuroendocrine cells. CgA/B are incorporated into immature vesicles that also contain other protein products destined for immature secretory vesicles (ISG) where chromogranins condense and bind to low-affinity receptors within the vesicle membrane. The development of ISGs into mature secretory granules (MSG) involves calcium (Ca2+) influx, granule acidification and prohormone processing and uptake of amines. Thereafter MSGs are directed to the plasma membrane and, after receptor-mediated Ca2+ influx, dock at the cell membrane. Docking is a complex process involving expression of syntaxin (SY), synaptotagmin (ST), VAMP2 (V2), and SNAP25 (S25). After docking, the MSG releases its contents into the extracellular fluid where their biological effects are transduced.
Chromogranin A—Biological Function and Clinical Utility in Neuro Endocrine Tumor Disease, 2010.
Extracellular functions of granins.
Granins serve as a precursor proteins that can be proteolitically processed by prohormone or proprotein convertases at multiple clivage sites to produce a large number of small bioactive peptides. More studies highlight the importance function for CgA in controlling both endocrine and neuroendocrine (NE) systems in vertebrates, but the in vivo endocrine function of these peptides remains unclear and current knowledge of chromogranin and related peptide physiology predominantly reflects in vitro cell culture studies.
Regulation of Parathormone Secretion. High plasma calcium levels and CgA-derivated peptides activate the calcium sensor. Vasostatin I is located in the parathyroid granules and in cosecreted with CgA at low plasma calcium levels, providing an autoinhibitory effect on parathormone secretion.
Processing of chromogranin A in the parathyroid: generation of parastatin-related peptides, 2000.
Regulation of Carbohydrate and Lipid Metabolism. Betagranin. N-terminal CgA fragment, is colocalized with insulin in the secretory granules and potentialy inhibits insulin secretion from islet cells in response to both glucose and potassium, by blocking calcium influx.
Pancreastatin inhibition of glucose-induced insulin release is mediated via an effect on G-protein and calcium-mediated exocytosis. In humans with diabetes is elevated and in vivo infusion of pancreastatin decreases glucose uptake.
Regulation on Catecholamine secretion. Nicotinic cholinergic receptor, when activated, triggers 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 releasing Catestatin, can exerts potent antagonistic effects on nicotinic cholinergic signaling, resulting in negative-feedback modulation of catecholamine release.
Regulation of Cardiovascular Function. Vasostatins, released by sympathetic nerve terminals, inhibit arterial vasoconstriction, TNF-α and the proangiogenetic activity of vascular endothelial growth factor (VEGF).
The cardioinhibitory effects of catestatin suggest that it also acts as a cardiac modulator by protecting the heart against sympathetic over activation. In humans, CgA levels correlate positively with endothelin-1 and negatively with glomerular filtration rate, suggesting that CgA may act through the glomerular endothelium to regulate renal function.
Regulation of Inflammatory Responses. Increased CgA levels correlate with serum TNF-α and/or soluble TNF-α receptor levels in a number of inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, chronic heart failure, and chronic obstructive pulmonary disease.
Circulating chromogranin A reveals extra-articular involvement in patients with rheumatoid arthritis and curbs TNF-alpha-elicited endothelial activation, 2008.
Regulation of Reproduction. The role of granins and their derived peptides in the control of vertebrate reproduction remains largely unexplored.
Chromogranin A—Biological Function and Clinical Utility in Neuro Endocrine Tumor Disease, 2010.
CGA LEVEL IN HEALTH AND DISEASE.
Level of CgA has a mean day-to-day variation of 25%. Food intake can also increase CgA levels.
Neoplastic conditions.
More important are CgA concentrations in NET, tumors that origin from NE cells (NE cells). NE cells are particular cells that could release their products in blood stream like endocrine cells, but have common characteristics and markers with neural cells.
It is possible to identify NE cells in many different organs and/or tissues such as: thyroid cells, pancreatic islet cells, bronchopulmonary tree, thymic endocrine cells, biliary tract and liver endrocrine cells, gastrointestinal endocrine cells, Merkel cells in skin, parathyroid cells, hypophysis, adrenal medulla and endocrine cells in other sites.
Pancreas stained with anti-Chromogranin A
The NE cells that give rise to NETs have many common features but are also unique in that each secretes a variety of bioactive products that defines its precise regulatory role. Thus, individual tumors are characterized by both a common biological signature related to their neuroendocrine role, as well as a specific biochemical signature unique to their precise function.
CgA concentrations are sensitive but not specific markers of NETs. CgA may also be regarded as an early marker of NETs of the foregut and midgut, with a specifity and sensivity of respectively 86% and 68%.
CgB and CgC are less sensitive indicators of NETs compared to CgA.
Some studies have reported an association between CgA and tumor location or degree of determination. Ileal NETs and GEP-NETs in MEN-1 had the highest values, meanwhile gastric type I, pituitary and parathyroid tumors had lower values.
Most well-differentiated NET cells intensely express CgA, like their normal NE cells counterparts. Conversely, poorly differentiated NE carcinomas rarely express CgA.
Chromogranin A—Biological Function and Clinical Utility in Neuro Endocrine Tumor Disease, 2010.
Several non-NE carcinomas exhibit patters of NE differentiation, and some of these tumors have elevated serum-CgA level. An example is prostate cancer. The incidence of NE cells in prostatic adenocarcinomas ranges from 10% to 100% and a positive correlation of CgA-positive NE tumor cells with serum level of CgA has been described. Elevated CgA seems to indicate a poor prognosis.
Chromogranin A and biochemical progression-free survival in prostate adenocarcinomas submitted to radical prostatectomy, 2007.
As shown in Figure 7 there are also “mixed exocrine-endocrine carcinoma” or “adenocarcinoma with NE differentiation”. The morphological, immunophenotypical and terminological heterogeneity in these tumors has obtained limited clinical attention, mainly due to the low prognostic impact of NE differentiation in conventional (adeno)carcinomas. Diagnostic parameters for these tumor, proposed by WHO, are:
1. extension of each component (at least 30%)
2. structural features of the NE components as well-differentiated organoid or solid/diffuse growth patterns.
Immunoreactivity for chromogranin A is generally the easiest and commonest procedure, which allows to detect NE differentiation.
The grey zone between pure (neuro)endocrine and non-(neuro)endocrine tumours: a comment on concepts and classification of mixed exocrine–endocrine neoplasms, 2006.
Non neoplastic conditions.
CgA elevation may caused also by non-neoplastic conditions:
- Renal failure: CgA increases in proportion of renal insufficiency due to decreased plasma clearance
- Autoimmune chronic atrophic gastritis: chronic hypergastrinemia and ECL cell proliferation, due to parietal cell damage, lead to increase in circulating CgA level
- Acid secretory medication such as PPIs
- Essential hypertension and chronic heart failure
- Hypertiroidism is associated with elevation of CgA in association with free T3 and free T4 level
- Liver cirrhosis, chronic hepatitis, pancreatitis, inflammatory bowel disease.
Chromogranin A and biochemical progression-free survival in prostate adenocarcinomas submitted to radical prostatectomy, 2007.
CONCLUSIONS.
CgA is currently the best available biomarker for the diagnosis of NETs. A critical issue is late diagnosis due to failure to identify symptoms or to establish the biochemical diagnosis. Early diagnosis requires attention.
The development of a variety of sensitive and specific plasma (P) and/or serum (S) assays for peptides and amines produced by these tumors and the development of immunohistochemistry panels has facilitated both blood and tissue diagnosis.
Measurements of serum and plasma CgA concentrations in patients with NETs reflect the degree of NE differentiation, total tumor burden and the effect of medical treatment.
Several different CgA antibodies are commercially available and post-translational processing of CgA is more extensive in NETs compared with normal NE cells. Regular (diffuse) strongly positive CgA immunoreactivity was predominant in typical NETs, whereas atypical tumors were characterized by irregular, patchy staining, probably reflecting the degree of maturation (functional state) of NE granules within the neoplastic cells.
