Serpinin: A Novel Chromogranin A-Derived, Secreted Peptide Up-Regulates Protease Nexin-1 Expression and Granule Biogenesis in Endocrine Cells

ORIGINAL RESEARCH Serpinin: A Novel Chromogranin A-Derived, Secreted Peptide Up-Regulates Protease Nexin-1 Expression and Granule Biogenesis in Endocrine Cells Hisatsugu Koshimizu, Niamh X. Cawley, Taeyoon Kim, Alfred L. Yergey, and Y. Peng Loh Section on Cellular Neurobiology (H.K., N.X.C., T.K., Y.P.L.), Program on Developmental Neuroscience, and Section on Metabolic Analysis and Mass Spectrometry (A.L.Y.), Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nM serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6 –22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules. (Molecular Endocrinology 25: 732–744, 2011) ense-core secretory granules (DCG) are required for the regulated secretion of peptide hormones from endocrine and neuroendocrine cells. Biogenesis of DCG is a critical step in the processing, storage, and sequestration of peptide hormones (1–3). Chromogranins (Cg), which are secreted with catecholamines from DCG upon stimulation, were the first granins to be identified in chromaffin cells (4). CgA, CgB, and secretogranin II–IV, belong to the D granin family and are abundantly expressed and stored in DCG in endocrine and neuroendocrine cells. In the granulogenic protein model, the granins aggregate to each other to form the dense core of secretory granules. CgA and CgB are able to induce DCG-like structures when overexpressed in fibroblasts, as does overexpression of prohormones/proneuropeptides, such as proopiomelanocortin, provasopressin, and prooxytocin (5, 6). ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/me.2010-0124 Received April 5, 2010. Accepted February 28, 2011. First Published Online March 24, 2011 Abbreviations: ActD, Actinomycin D; 8-Br-cAMP, 8-bromo-cAMP; Cg, chromogranin; CREB, cAMP response element-binding protein; DCG, dense-core secretory granule; EIA, enzyme immunoassay; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; KO, knock-out; MALDI-TOF, matrix assisted laser desorption/ionization time of flight; MitA, mithramycin A; MS/MS, tandem mass spectrometry; PC, prohormone convertase; pCREB, phosphorylated cAMP response element-binding protein; PKA, protein kinase A; PN-1, protease nexin 1; RRG, argininearginine-glycine; serpin, serine protease inhibitor; 6T3-WT, 6T3 wild-type; TFA, trifluoroacetic acid. 732 mend.endojournals.org Mol Endocrinol, May 2011, 25(5):732–744 Mol Endocrinol, May 2011, 25(5):732–744 Our previous studies demonstrated that CgA plays a critical role in augmenting DCG biogenesis in (neuro)endocrine cells using a novel mechanism. We found that down-regulation of CgA expression impaired DCG formation and activity-dependent secretion of an exogenous cargo molecule, proopiomelanocortin, transfected into rat adrenal pheochromocytoma cell line, PC12 cells (6). However, in a mutant PC12 cell line, PC12–27, which has permanently lost the regulated-secretion programs at the genetic level, CgA was unable to rescue DCG formation, indicating that CgA cannot activate genetic programs that drive granule biogenesis (7, 8). We have also reported that exogenous expression of bovine CgA in 6T3 wild-type (6T3-WT) cells, a variant of the AtT-20 mouse pituitary cell line, which lacks CgA and DCG, induced de novo DCG biogenesis and restored regulated secretion in these cells (6). In one study, CgA-knockout (KO) mice showed a significant decrease in the size and number of DCG in adrenal chromaffin cells (9). However, another CgA-KO mouse generated by Hendy et al. (10) showed no significant differences in the numbers of chromaffin granules compared with wild-type mice. In addition to the CgA-KO mice, CgA-antisense transgenic mice deficient in CgA have been shown to have significantly decreased numbers of chromaffin granules (11). Hence several studies thus far indicate that CgA plays a major role in DCG biogenesis both in vitro and in vivo. Subsequent DNA microarray analysis carried out on 6T3-WT cells with and without transfection of bovine CgA revealed that the mechanism of action of CgA in granule biogenesis involved elevation of mRNA of a serine protease inhibitor (serpin) family serine protease inhibitor, protease nexin-1 (PN-1) (12). PN-1 was shown to be localized to the Golgi apparatus and functioned to stabilize the levels of DCG proteins necessary for the DCG biogenesis (12). Thus, in addition to its granulogenic function, CgA has been shown to augment DCG biogenesis by up-regulating PN-1 expression. However, the molecular mechanism by which CgA signals the up-regulation of PN-1 expression remains unclear. In this study, we investigated the nature of the CgArelated molecule that mediates the up-regulation of PN-1 expression in 6T3-WT cells and PC12 cells. We synthesized several CgA-derived peptides based on paired basic residue sites where proteolytic cleavage takes place and identified one of less than 3 kDa in size, which represents the C terminus of CgA (13) that was able to up-regulate PN-1 mRNA expression in 6T3-WT cells and PC12 cells. This peptide, which we named serpinin, was subsequently shown to be present endogenously in AtT-20 cells (...truncated)