Chromogranin A in the early steps of the neurosecretory pathway.

Chromogranin A (CgA) is a soluble glycoprotein stored with hormones and neuropeptides in secretory granules (SG) of most (neuro)endocrine cells and neurons. Since its discovery in 1967, many studies have reported its structural characteristics, biological roles, and mechanisms of action. Indeed, CgA is both a precursor of various biologically active peptides and a granulogenic protein regulating the storage and secretion of hormones and neuropeptides. This review emphasizes the findings and theoretical concepts around the CgA-linked molecular machinery controlling hormone/neuropeptide aggregation and the interaction of CgA-hormone/neuropeptide aggregates with the trans-Golgi membrane to allow hormone/neuropeptide targeting and SG biogenesis. We will also discuss the intriguing alteration of CgA expression and secretion in various neurological disorders, which could provide insights to elucidate the molecular mechanisms underlying these pathophysiological conditions.

Chromogranin A and its fragments in cardiovascular, immunometabolic, and cancer regulation.

Chromogranin A (CgA)-the index member of the chromogranin/secretogranin secretory protein family-is ubiquitously distributed in endocrine, neuroendocrine, and immune cells. Elevated levels of CgA-related polypeptides, consisting of full-length molecules and fragments, are detected in the blood of patients suffering from neuroendocrine tumors, heart failure, renal failure, hypertension, rheumatoid arthritis, and inflammatory bowel disease. Full-length CgA and various CgA-derived peptides, including vasostatin-1, pancreastatin, catestatin, and serpinin, are expressed at different relative levels in normal and pathological conditions and exert diverse, and sometime opposite, biological functions. For example, CgA is overexpressed in genetic hypertension, whereas catestatin is diminished. In rodents, the administration of catestatin decreases hypertension, cardiac contractility, obesity, atherosclerosis, and inflammation, and it improves insulin sensitivity. By contrast, pancreastatin is elevated in diabetic patients, and the administration of this peptide to obese mice decreases insulin sensitivity and increases inflammation. CgA and the N-terminal fragment of vasostatin-1 can enhance the endothelial barrier function, exert antiangiogenic effects, and inhibit tumor growth in animal models, whereas CgA fragments lacking the CgA C-terminal region promote angiogenesis and tumor growth. Overall, the CgA system, consisting of full-length CgA and its fragments, is emerging as an important and complex player in cardiovascular, immunometabolic, and cancer regulation.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 µM) and PACAP (0.1 µM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 µM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

Catestatin: A Master Regulator of Cardiovascular Functions.

BACKGROUND: Cardiovascular disease (CVD), the most common cause of death globally, accounts for ~30% of all deaths worldwide. Hypertension is a common contributor to morbidity and mortality from CVD. METHODS AND RESULTS: The plasma concentration of chromogranin A (CgA) is elevated in patients with CVD as well as patients with established human essential hypertension and heart failure (HF). In contrast, the plasma level of the CgA-derived peptide catestatin (CST) is diminished in human essential hypertension. Low conversion of CgA-to-CST has been associated with increased mortality in patients hospitalized with acute HF. Consistent with human findings, the lack of CST in CgA knockout (Chga-KO) mice eventuates in the development of hypertension and supplementation of CST to Chga-KO mice restores blood pressure, implicating CST as a key player in regulating hypertension. In the peripheral system, CST decreases blood pressure by stimulating histamine release, inhibiting catecholamine secretion, or causing vasodilation. Centrally, CST improves baroreflex sensitivity (BRS) and heart rate variability (HRV) by exciting GABAergic neurons in the caudal ventrolateral medulla (CVLM) and pyramidal neurons of the central amygdala; CST also decreases BRS by exciting glutamatergic rostral ventrolateral medulla (RVLM) neurons. In addition, CST provides cardioprotection by inhibiting inotropy and lusitropy; activating mitochondrial KATP channels, and stimulating reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways and consequent inhibition of mitochondrial permeability transition pore (mPTP). CST modulates cardiomyocyte Ca2+ levels by direct inhibition of Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) activity and consequent reduction in phosphorylation of phospholamban and ryanodine receptor 2, thereby providing support for a direct functional role of CST in the failing myocardium. CONCLUSION: These multitude of effects establish CST as a master regulator of cardiovascular functions.

Dense-core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B.

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense-core vesicle (DCV) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY)-pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT-PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B-/- neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B-/- neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY-pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH-sensitive NPY-pHluorin fluorescence, were all normal in CgA/B-/- neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B-/- neurons. In contrast, CgA/B-/- chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live-cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B-/- neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

Role of the neuroendocrine antimicrobial peptide catestatin in innate immunity and pain.

Catestatin (CST) is a neuroendocrine peptide which is derived from the chromogranin A. It has been demonstrated that CST can affect a wide range of processes, such as innate immunity, inflammatory and autoimmune reactions, and several homeostatic regulations. Furthermore, CST is positive against several kinds of bacterial strains at micromolecular range, which shows its antimicrobial activity. Recently, the role of CST in acute and chronic pain has attracted much attention. In this review, we discussed the latest research findings of CST and its role in innate immunity and pain.

Chromogranin A regulates vesicle storage and mitochondrial dynamics to influence insulin secretion.

Chromogranin A (CgA) is a prohormone and a granulogenic factor that regulates secretory pathways in neuroendocrine tissues. In ß-cells of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. The impact of CgA deficiency on the formation and exocytosis of insulin vesicles is yet to be investigated. In addition, no literature exists on the impact of CgA on mitochondrial function in ß-cells. Using three different antibodies, we demonstrate that CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. CgA deficiency in Chga-KO islets leads to compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins and insulin genes, as well as increased insulin protein content. Ultrastructural studies of pancreatic islets revealed that Chga-KO ß-cells contain fewer immature secretory granules than wild-type (WT) control but increased numbers of mature secretory granules and plasma membrane-docked vesicles. Compared to WT control, CgA-deficient ß-cells exhibited increases in mitochondrial volume, numerical densities and fusion, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1 and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion observed in Chga-KO mice. We conclude that CgA is an important regulator for coordination of mitochondrial dynamics, secretory vesicular quanta and GSIS for optimal secretory functioning of ß-cells, suggesting a strong, CgA-dependent positive link between mitochondrial fusion and GSIS.

A Critical Approach to Clinical Biochemistry of Chromogranin A.

Chromogranin A (CGA) is a member of the granin family of proteins which are widespread in endocrine, neuroendocrine, peripheral, and central nervous tissues, where they are typically found in secretory granules. It is well accepted that CGA cooperates to regulate synthesis and secretion of these various granule signaling molecules.Because of its ubiquitous distribution within neuroendocrine tissues, CGA can be a useful diagnostic marker for neuroendocrine neoplasms, including carcinoids, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas (MTC), some pituitary tumors, functioning and nonfunctioning islet cell tumors and other amine precursor uptake and decarboxylation (APUD) tumors. It is also useful as a prognostic marker for detection of recurrence and monitoring of response to different treatments. As other tumor markers, it is imperative to know its physiology and pathophysiology, its sensitivity and specificity in different neuroendocrine tumors (NETs), and carefully integrate these data with the clinical data of the single patient, to maximize its diagnostic/prognostic index.

Chromogranin A: a paradoxical player in angiogenesis and vascular biology.

Half a century after the discovery of chromogranin A as a secreted product of the catecholamine storage granules in the bovine adrenal medulla, the physiological role for the circulating pool of this protein has been recently coined, namely as an important player in vascular homeostasis. While the circulating chromogranin A since 1984 has proved to be a significant and useful marker of a wide range of pathophysiological and pathological conditions involving the diffuse neuroendocrine system, this protein has now been assigned a physiological "raison d'etre" as a regulator in vascular homeostasis. Moreover, chromogranin A processing in response to tissue damage and blood coagulation provides the first indication of a difference in time frame of the regulation of angiogenesis evoked by the intact chromogranin A and its two major peptide products, vasostatin-1 and catestatin. The impact of these discoveries on vascular homeostasis, angiogenesis, cancer, tissue repair and cardio-regulation will be discussed.

The important role of catestatin in cardiac remodeling.

Catestatin (CST) was first discovered as a potent non-competitive and reversible inhibitor of catecholamine secretion. Recent reports on plasma CST level in heart diseases suggested a cardioprotective role for this peptide. Given that cardiac remodeling is the dominant pathologic process in cardiac dysfunction, we propose that CST participates in the regulation of concern pathways and contributes to the inhibition of cardiac remodeling. In this minireview, the potential mechanism of cardiac remodeling involving CST will be discussed from three aspects: hypertrophy, fibrosis, and apoptosis.

Granins and catecholamines: functional interaction in chromaffin cells and adipose tissue.

Catecholamines (CAs) and granin peptides are costored in dense-core vesicles within the chromaffin cells of the adrenal medulla and in other endocrine organs and neurons. Granins play a major functional and structural role in chromaffin cells but are ubiquitous proteins, which are present also in secretory cells of the nervous, endocrine, and immune systems, where they regulate a number of cellular functions. Furthermore, recent studies also demonstrate that granin-derived peptides can functionally interact with CA to modulate key physiological functions such as lipolysis and blood pressure. In this chapter, we will provide a brief update on the interaction between CA and granins at the cellular and organ levels. We will first discuss recent data on the regulation of exocytosis of CA and peptides from the chromaffin cells by the sympathetic nervous system with a specific reference to the prominent role played by splanchnic nerve-derived pituitary adenylate cyclase-activating peptide (PACAP). Secondly, we will discuss the role of granins in the storage and regulation of exocytosis in large dense-core vesicles. Finally, we will provide an up-to-date review of the roles played by two granin-derived peptides, the chromogranin A-derived peptide catestatin and the VGF-derived peptide TLQP-21, on lipolysis and obesity. In conclusion, the knowledge gathered from recent findings on the role played by proteins/peptides in the sympathetic/target cell synapses, discussed in this chapter, would contribute to and provide novel mechanistic support for an increased appreciation of the physiological role of CA in human pathophysiology.

Differential pathways for interleukin-1ß production activated by chromogranin A and amyloid ß in microglia.

Although chromogranin A (CGA) is frequently present in Alzheimer's disease (AD), senile plaques associated with microglial activation, little is known about basic difference between CGA and fibrillar amyloid-ß (fAß) as neuroinflammatory factors. Here we have compared the interleukin-1ß (IL-1ß) production pathways by CGA and fAß in microglia. In cultured microglia, production of IL-1ß was induced by CGA, but not by fAß. CGA activated both nuclear factor-κB (NF-κB) and pro-caspase-1, whereas fAß activated pro-caspase-1 only. For the activation of pro-caspase-1, both CGA and fAß needed the enzymatic activity of cathepsin B (CatB), but only fAß required cytosolic leakage of CatB and the NLRP3 inflammasome activation. In contrast, fAß induced the IL-1ß secretion from microglia isolated from the aged mouse brain. In AD brain, highly activated microglia, which showed intense immunoreactivity for CatB and IL-1ß, surrounded CGA-positive plaques more frequently than Aß-positive plaques. These observations indicate differential pathways for the microglial IL-1ß production by CGA and fAß, which may aid in better understanding of the pathological significance of neuroinflammation in AD.

[Chromogranin A and neuroendocrine tumors]. / Cromogranina A y tumores neuroendocrinos.

Chromogranin A (CgA) is the most abundant granin in gastroenteropancreatic neuroendocrine tumors (GEP-NETs). As a tumor marker is moderately sensitive and nonspecific. Despite the limitations of testing methods, which require careful interpretation, especially in the case of gastrinomas, patients treated with somatostatin analogues, and poorly differentiated tumors, it is the best tumor marker in GEP-NETs and may be of value in other tumors with neuroendocrine differentiation. CgA may be used as a marker in blood or tissue samples through immunohistochemical techniques. CgA levels correlate with tumor burden and extension and may be used for diagnosis and monitoring of GEP-NETs, especially midgut carcinoids and endocrine pancreatic tumors. It is also useful as a prognostic marker for detection of recurrence and monitoring of response to different treatments.

The functional role of chromogranins in exocytosis.

Chromogranins A (CgA) and B (CgB) are the main soluble proteins of large dense-core secretory vesicles (LDCVs). Using CgA- and CgB-knockout (KO) mice, we found that the absence of chromogranins A and B induces significant changes in catecholamine (CA) accumulation and the kinetics of exocytosis. By crossing these two knockout strains, we generated a viable and fertile double CgA/B-KO mouse in which the catecholamine content in chromaffin LDCVs was halved, and the secretory response significantly reduced. Incubating cells with L-DOPA increased the vesicular CA content in wild-type (WT) but not in Cg-KO cells, which was not due to changes in amine transport, or in the synthesis or degradation of cytosolic amines. Electron microscopy revealed the presence of giant secretory vesicles exhibiting significant alterations, with little or no electrodense inner matrix. Proteomic analysis confirmed the absence of CgA and B, and revealed small changes in SgII in the LDCV-enriched fraction, as well as the overexpression of fibrinogen and other proteins. In summary, our findings indicate that the mechanisms responsible for vesicular accumulation of CA are saturated in Cgs-KO cells, in contrast to the ample capacity for further accumulation in WT cells. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and exocytosis in LDCVs.

Chromogranin A and derived peptides in health and disease.

Chromogranin A (CgA) is a member of the granins, a family of acidic proteins found in abundance in (neuro)endocrine cells (e.g., in chromaffin cells) and in some tumors. Like other granins, CgA has a granulogenic role in secretory granule biogenesis and is stored in these organelles. CgA is partially processed differentially in various cell types to yield biologically active peptides, such as vasostatin, pancreastatin, catestatin, and serpinins. In this review, we describe the roles of CgA and several of its derived peptides. CgA, which is elevated in the blood of cancer patients, inhibits angiogenesis and exerts protective effects on the endothelial barrier function in tumors, thus affecting response to chemotherapy. Recent studies indicate that the serpinins promote cell survival and myocardial contractility and relaxation. Other peptides such as pancreastatin were found to have significant effects on inhibition of glucose-stimulated insulin secretion and glucose up-take, induction of glycogenolysis in hepatocytes, and inhibition of lipogenesis. In contrast, catestatin has opposite effects to that of pancreastatin in glucose metabolism and lipogenesis. Catestatin appears to also play a significant role in cardiac function, blood pressure regulation, and mutations in the catestatin domain of the CgA gene are associated with hypertension in humans.

The novel chromogranin A-derived serpinin and pyroglutaminated serpinin peptides are positive cardiac ß-adrenergic-like inotropes.

Three forms of serpinin peptides, serpinin (Ala26Leu), pyroglutaminated (pGlu)-serpinin (pGlu23Leu), and serpinin-Arg-Arg-Gly (Ala29Gly), are derived from cleavage at pairs of basic residues in the highly conserved C terminus of chromogranin A (CgA). Serpinin induces PN-1 expression in neuroendocrine cells to up-regulate granule biogenesis via a cAMP-protein kinase A-Sp1 pathway, while pGlu-serpinin inhibits cell death. The aim of this study was to test the hypothesis that serpinin peptides are produced in the heart and act as novel ß-adrenergic-like cardiac modulators. We detected serpinin peptides in the rat heart by HPLC and ELISA methods. The peptides included predominantly Ala29Gly and pGlu-serpinin and a small amount of serpinin. Using the Langendorff perfused rat heart to evaluate the hemodynamic changes, we found that serpinin and pGlu-serpinin exert dose-dependent positive inotropic and lusitropic effects at 11-165 nM, within the first 5 min after administration. The pGlu-serpinin-induced contractility is more potent than that of serpinin, starting from 1 nM. Using the isolated rat papillary muscle preparation to measure contractility in terms of tension development and muscle length, we further corroborated the pGlu-serpinin-induced positive inotropism. Ala29Gly was unable to affect myocardial performance. Both pGlu-serpinin and serpinin act through a ß1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway, indicating that, contrary to the ß-blocking profile of the other CgA-derived cardiosuppressive peptides, vasostatin-1 and catestatin, these two C-terminal peptides act as ß-adrenergic-like agonists. In cardiac tissue extracts, pGlu-serpinin increased intracellular cAMP levels and phosphorylation of phospholamban (PLN)Ser16, ERK1/2, and GSK-3ß. Serpinin and pGlu-serpinin peptides emerge as novel ß-adrenergic inotropic and lusitropic modulators, suggesting that CgA and the other derived cardioactive peptides can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation.

Chromogranin A is a reliable biomarker for gastroenteropancreatic neuroendocrine tumors in an Asian population of patients.

PURPOSE: To evaluate the significance of plasma chromogranin A (CgA) levels in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NET) in terms of disease status and treatment responses. MATERIALS AND METHODS: Forty-four GEP-NET patients comprising 15 disease-free patients and 29 patients with active disease, as well as 26 healthy participants were enrolled in this study between April 2010 and April 2011. Clinicopathological factors were collected and serial plasma CgA levels were measured. RESULTS: Plasma CgA levels were significantly higher in GEP-NET patients with active disease than in disease-free patients (p = 0.011) or healthy participants (p = 0.001). No difference in CgA levels was observed in terms of primary tumor location, tumor grade, and functional status in patients with active disease. CgA values at 94 U/l distinguished healthy individuals or disease-free patients from patients with active disease. Sensitivity and specificity rates were 86 and 88%, respectively. CgA levels at 110 U/l differentiated patients without recurrence from those with recurrence, with a sensitivity rate of 100% and a specificity rate of 80%. Patients (5/5, 100%) with stable disease and who showed partial response after treatment had a more than 20% decrease in CgA levels compared with the baseline values. Patients (6/6, 100%) with progressive disease showed a less than 20% decrease or increase in CgA levels. CONCLUSION: The plasma CgA level is a reliable biomarker for GEP-NET. We conclude that changes in CgA levels are associated with disease status and treatment responses.

Determination of the lactate threshold by means of salivary biomarkers: chromogranin A as novel marker of exercise intensity.

This study examined intra-individual variations in salivary lactate (sLac), alpha-amylase (sAA) and chromogranin A (sCgA) with reference to the accumulation of blood lactate (bLac) during incremental maximal exercise in swimmers. Samples of blood and saliva were collected simultaneously from 12 male professional athletes during an incremental test that consisted of eight series of 100 m in front crawl with increasing velocity (0.03 m s(-1) each) and 70-s intervals. The concentration of blood and salivary lactate was determined by an electro-enzymatic assay, whereas sAA and CgA were analysed by Western blotting. Inflection points in the concentration of bLAc, sLac, sAA and CgA were found in all subjects. The accumulation of lactate in saliva followed the same pattern observed in blood with a high correlation between the two (r = 0.91). Similar results were observed between the dynamics of sAA (r = 0.81) and sCgA (r = 0.82) in relation to bLac. These findings support the usefulness of saliva for the determination of the lactate threshold and provide the first demonstration of sCgA as a novel marker of exercise intensity in well-trained men.

The vasostatin-1 fragment of chromogranin A preserves a quiescent phenotype in hypoxia-driven endothelial cells and regulates tumor neovascularization.

The angiogenic switch is a fundamental process for many diseases and for tumor growth. The main proangiogenic stimulus is hypoxia, through activation of the hypoxia-inducible factor (HIF)-1α pathway in endothelial cells (ECs). We have previously shown that the vasostatin-1 (VS-1) fragment of chromogranin A inhibits TNF-α-induced vessel permeability and VEGF-induced EC proliferation, together with migration and matrix invasion, which are all critical steps in angiogenesis. The present study was undertaken to investigate the effect of VS-1 on tumor angiogenesis. We found mouse mammary adenocarcinomas (TS/A), genetically engineered to secrete VS-1 (TS/A 1B8), to be characterized by reduced vascular density and more regular vessels, compared with nontransfected tumors [TS/A wild type (WT)]. Supernatants from TS/A WT cells, but not those from TS/A 1B8, generated tip cells and promoted the permeability of primary human umbilical vein ECs, via VE-cadherin redistribution and cytoskeletal disorganization. These effects were abrogated by mAb 5A8, a VS-1-blocking antibody. Furthermore, VS-1 inhibited hypoxia-driven EC morphological changes, VE-cadherin redistribution, intercellular gap formation, tube morphogenesis, and HIF-1α nuclear translocation in vitro. Our findings highlight a previously undescribed function of VS-1 as a regulator of tumor vascularization.

Human catestatin enhances migration and proliferation of normal human epidermal keratinocytes.

BACKGROUND: Skin-derived antimicrobial peptides, such as human ß-defensins and cathelicidins, actively contribute to host defense by inactivating microorganisms. Catestatin, a neuroendocrine peptide that affects human autonomic functions, has recently been detected in keratinocytes upon injury/infection where it inhibits the growth of pathogens. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu, and Arg374Gln. OBJECTIVE: To investigate the effects of human catestatin and its variants on keratinocyte migration and proliferation, and to elucidate the possible signaling mechanisms involved. METHODS: The migration of normal human keratinocytes was analyzed using Boyden microchamber assay and in vitro wound closure assay. Cell proliferation was evaluated by BrdU incorporation, cell count assay and cell cycle analysis. Intracellular Ca(2+) mobilization was measured using a fluorescent calcium assay kit. The phosphorylation of epidermal growth factor receptor (EGFR), Akt, and MAPKs was determined by Western blotting. RESULTS: Catestatin and its variants dose-dependently enhanced keratinocyte migration and proliferation. Moreover, catestatin peptides increased intracellular Ca(2+) mobilization and induced the phosphorylation of EGFR, Akt, extracellular signal-regulated kinase (ERK), and p38 in keratinocytes. The induction of keratinocyte migration and proliferation by catestatin peptides involved G-proteins, phospholipase C, EGFR, PI3-kinase, ERK, and p38, as evidenced by the specific inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor), AG1478 (EGFR inhibitor), anti-EGFR antibody, wortmannin (PI3-kinase inhibitor), U0126 (ERK inhibitor), and SB203580 (p38 inhibitor), respectively. CONCLUSION: Besides inhibiting the growth of skin pathogens, catestatin peptides may also contribute to cutaneous wound closure by enhancing keratinocyte migration and proliferation at the wound site.