Secretogranin II Gene Research Articles

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins (R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression (R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca(2+) concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

3′, 5′-Cyclic Adenosine 5′-Monophosphate Response Element-Dependent Transcriptional Regulation of the Secretogranin II Gene Promoter Depends on Gonadotropin-Releasing Hormone-Induced Mitogen-Activated Protein Kinase Activation and the Transactivator Activating Transcription Factor 3

Previous studies demonstrated that GnRH-induced secretogranin II (SgII) promoter regulation required a consensus cAMP response element (CRE) and protein kinase A/CRE binding protein. The present studies examined the role of additional components of the GnRH signaling network on SgII promoter activity with particular attention devoted to CRE-dependent gene regulation. Disruption of the SgII CRE by mutagenesis resulted in inhibition of GnRH agonist (GnRHa) induction of this promoter in alphaT3-1 cells. Pharmacological and dominant-negative inhibition of the ERK and c-Jun N-terminal kinase (JNK) signaling pathways revealed that GnRHa-induced SgII promoter activity required functional JNK and ERK modules. Combined inhibition of both pathways nearly abolished GnRHa-induced SgII promoter activity. Specific induction of the ERK cascade alone using overexpression of Raf-CAAX was not sufficient to activate the SgII gene promoter. In contrast, overexpression of the catalytic domain of the more pleiotropic MAPK activator, MAPK/ERK kinase-1, was sufficient to induce SgII promoter activity. The effect(s) of mitogen-activated protein/ERK kinase-1 on SgII promoter activity was CRE dependent and was reversed by the combined pharmacological inhibition of both JNK and ERK modules. CRE DNA binding studies demonstrated the recruitment of activating transcription factor (ATF)-3 and c-Jun to the CRE after administration of GnRHa to alphaT3-1 cells. Specific small interfering RNA knockdown of ATF3 reduced ATF3 DNA binding and the effect of GnRHa on the SgII promoter. These studies support the conclusion that MAPK signaling and ATF3 action are essential for full SgII promoter activation by GnRHa through a canonical CRE. Moreover, we suggest that within the GnRH signaling network, CRE-dependent gene regulation in general may be mediated primarily through the immediate early response gene ATF3.

Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells

Secretoneurin (SN) and EM66 are two highly conserved peptides that derive from the processing of secretogranin II (SgII), one of the major constituents of chromaffin cell secretory vesicles. It has been shown that PACAP regulates SgII gene transcription and SN release in bovine adrenochromaffin cells. The aim of the present study was to localize and characterize EM66 in the bovine adrenal gland, and to examine the signaling pathways activated by PACAP to regulate the secretion of EM66 from cultured chromaffin cells. Double immunohistochemical labeling showed an intense EM66-immunoreactive (EM66-IR) signal in TH-positive medullary chromaffin cells of the adrenal gland. HPLC analysis combined with RIA detection revealed, in adrenal medulla extracts and cultured chromaffin cell media, the presence of a major EM66-IR peak co-eluting with the recombinant peptide. PACAP dose-dependently stimulated EM66 release from chromaffin cells (ED 50 = 4.8 nM). The effect of PACAP on EM66 secretion was observed after 6 h of treatment and increased to reach a 2.6-fold stimulation at 48 h. The nonselective calcium channel blocker NiCl 2, the cytosolic calcium chelator BAPTA-AM and the L-type calcium channel blocker nimodipine significantly inhibited the stimulatory effect of PACAP on EM66 release. The secretory response to PACAP was also significantly lowered by the protein kinase A inhibitor H89 and by the protein kinase C inhibitor chelerythrine. Concomitant administration of chelerythrine, H89, NiCl 2 and BAPTA totally abolished PACAP-stimulated EM66 secretion. The MAPK inhibitors U0126 and SB203580 respectively decreased by 63% and 72% PACAP-evoked EM66 release. These results indicate that, in bovine adrenal medulla, SgII is processed to generate the EM66 peptide and that PACAP activates multiple signaling pathways to regulate EM66 release from chromaffin cells, suggesting that EM66 may act downstream of the trans-synaptic stimulation of the adrenal medulla by neurocrine factors.

Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors.

Secretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by proteolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC(50) approximately 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroendocrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl(2) equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator forskolin or cell-depolarization by high K(+) were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to trans-acting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein-1-related proteins. These data indicate that SN biosynthesis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla.

Isolation and characterization of the human secretogranin II gene promoter

The goal of this study was to isolate and functionally characterize the human secretogranin II (SgII) gene promoter. SgII is a member of the granin family of proteins which are selectively expressed in neurosecretory cells. The human SgII promoter contains a consensus TATA box and cyclic AMP response element (CRE) 35 and 74 bp upstream of the transcription start site, respectively, elements also found in the mouse and rat SgII gene promoters. Transfection studies showed that 869 bp of the human SgII promoter were sufficient to confer cell type-specific expression of an SgII promoter-luciferase reporter gene in neurosecretory PC-12, GH and BE(2)-M17 cells. The activity of the human SgII promoter was also compared in three N-type, human neuroblastoma cell lines [BE(2)-M17, SMS-KAN and SH-SY5Y], which differ markedly in the level of SgII expression. SgII promoter activities in the neuroblastoma cell lines correlated not only with the levels of SgII but also the levels of the cyclic AMP response element-binding protein CREB which were highest in BE(2)-M17 cells and lowest in SH-SY5Y cells. To establish that the activity of the human SgII promoter in these neuroblastoma cell lines is dependent on the level of CREB, rat CREB was overexpressed in SH-SY5Y cells. SgII promoter activity was up to 8-fold higher in SH-SY5Y cells overexpressing CREB. These results suggest that SgII expression is a marker for neuronal differentiation in human neuroblastoma cell lines and is dependent on the level of CREB expression.

Synergistic action of upstream elements and a promoter-proximal CRE is required for neuroendocrine cell-specific expression and second-messenger regulation of the gene encoding the human secretory protein secretogranin II

Secretogranin II (SgII) is a secretory polypeptide stored in large dense core vesicles of neuroendocrine and neuronal cells. In order to characterize the molecular mechanisms underlying the tissue-specific expression of the SgII gene and its regulation by second-messenger pathways in endocrine and neuronal cells, we have cloned and characterized the human SgII gene. Sequence analysis revealed the existence of numerous putative cis-regulatory elements in the SgII gene promoter, including a consensus cyclic AMP-responsive element (CRE). Constructs containing different portions of the human SgII promoter fused to the luciferase reporter were transfected in AtT-20, SH-SY5Y, LLC-PK1 or COS-7 cells. Northern blot analysis showed that the endogenous SgII gene is more highly expressed in AtT-20 cells than in SH-SY5Y cells, and not expressed at all in LLC-PK1 cells. Treatment by forskolin or 12- O-tetradecanoylphorbol-13-acetate (TPA) caused a 1.5- and 10-fold increase, respectively, in SgII mRNA levels in SH-SY5Y cells but not in AtT-20 cells. Transfection experiments revealed that 4 kb of the human SgII promoter is sufficient to impart cell-specific expression of the reporter gene in the four cell lines studied. Specifically, in AtT-20 cells, a positive element located between−1.38 and−4 kb, in addition to the CRE, is responsible for the high expression of the SgII gene. In SH-SY5Y cells, a negative element located between−0.66 and−1.4 kb represses the activating effect of the CRE leading to an overall lower activity of fusion genes in these cells compared to the activity in AtT-20 cells. Finally, the promoter activity was very low in LLC-PK1 and COS-7 cells. Forskolin and TPA stimulated the activity of a SgII-luciferase fusion gene in SH-SY5Y but not in AtT-20 cells. Disruption of the CRE abolished the stimulatory effect of forskolin and TPA. These data suggest that the basal activity of the human SgII gene relies on cell-specific trans-acting factors in addition to factors that bind to the CRE and show that the regulation of this gene by second messengers is cell-specific and requires an intact CRE.

Coactivation of secretogranin-II and BDNF genes mediated by calcium signals in mouse cerebellar granule cells

In primary culture of mouse cerebellar granule cells, the brain-derived neurotrophic factor (BDNF) gene is activated in an activity-dependent manner, accompanying Ca 2+ influx into neurons through voltage-dependent calcium channels (VDCCs). In this study, we investigated the inducibility of secretogranin-II (Sg-II) gene in terms of Ca 2+ signals evoked via VDCCs, by a comparison with BDNF and c- fos genes. Deprivation and subsequent induction of membrane depolarization by lowering and reelevating the extracellular concentration of potassium chloride (KCl), respectively, led to an decrease and then an increase in the Sg-II, BDNF and c- fos mRNA expression. The increase in Sg-II mRNA expression was detected as early as but was slower than that of BDNF one. The increase in Sg-II mRNA expression was induced depending upon the extracellular Ca 2+ and inhibited by nicardipine, indicating a requirement of Ca 2+ influx through VDCCs for the Sg-II as well as BDNF gene induction. Inhibition of de novo protein synthesis by cycloheximide did not affect the Sg-II induction. The response of Sg-II gene to the changes in extracellular KCl concentration was the same as that of BDNF but different from that of c- fos gene. Thus, Sg-II gene is coactivated with BDNF gene in response to the intracellular Ca 2+ signals evoked via Ca 2+ influx through VDCCs.

Polyethylene glycol-induced hypovolemia affects the expression of MCH mRNA, but not dynorphin or secretogranin II mRNAs, in the rat lateral hypothalamus

Two prominent neuron populations of the rat lateral hypothalamus express genes encoding respectively the prepromelanin-concentrating hormone (MCH) or dynorphin (DYN) and secretogranin II (SGII). Their roles remain hypothetical in mammals. In the present study, we examined the changes in MCH, DYN and SGII gene expression in dehydrated rats compared to controls. Dehydration was obtained by subcutaneous injection of polyethylene glycol (PEG) resulting in a large reduction of the extracellular fluid volume. Using competitive semi-quantitative RT-PCR and in situ hybridization methods, PEG-injected animals showed a significant increase of MCH mRNA level but no variation of DYN and SGII mRNA levels. These results confirm previous observations suggesting that intra- and extracellular dehydration challenges affect different regulation circuits; they indicate that both neuron populations could be involved in the maintenance of body fluid homeostasis, directly, or indirectly, as integrators of various information leading to goal-oriented behaviour.

Induction of secretogranin II mRNA by protein synthesis inhibitors in GH4C1 cells

The effect of cycloheximide (CHX) on the expression of secretogranin II (SgII), a member of the granin family of secretory proteins, was investigated in rat pituitary GH4C1 (GH) cells. The administration of CHX resulted in a dose- and time-dependent increase in SgII mRNA expression, the greatest effect (3.8-fold above control) being achieved with 1 microgram/ml CHX, which resulted in > 90% inhibition of protein synthesis. Emetine (1 microgram/ml), pactamycin (0.6 microgram/ml), anisomycin (2.5 micrograms/ml), and puromycin (100 micrograms/ml), protein synthesis inhibitors structurally and mechanistically unrelated to CHX, also increased the level of SgII mRNA expression. Treatment with forskolin (10 microM) alone had no effect on SgII mRNA levels but potentiated the effect of CHX. Neither 100 nM phorbol 12-myristate 13-acetate nor 45 mM KCl affected SgII mRNA levels in the absence or presence of 1 microgram/ml CHX. The effect of CHX was blocked by the transcription inhibitors actinomycin D (5 micrograms/ml) and 5,6-dichlorobenzimidazole riboside (20 micrograms/ml) but not by the coadministration of the polysome destabilizer pactamycin (0.6 microgram/ml), suggesting that the effect of CHX was transcriptional. These studies show that the expression of SgII mRNA is induced by protein synthesis inhibitors in GH cells, suggesting the presence of a labile repressor, which not only controls the basal expression of the SgII gene but also completely inhibits the stimulatory effect of forskolin.

Secretogranin II Expression in Ewing??s Sarcomas and Primitive Neuroectodermal Tumors

Production of chromogranins, the acidic components of the chromaffin granules regarded as specific neuroendocrine markers, was analyzed by immunocytochemistry and hybridization (Northern blotting and in situ hybridization) in primary lesions and cell lines of Ewing's sarcomas, primitive neuroectodermal tumors (PNETs), and neuroblastomas. Antibodies and probes specific for chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) were used. Ewing's sarcomas and PNETs, unlike neuroblastomas, were negative for CgA and CgB. Two primary Ewing's sarcomas, one primary PNET (an Askin tumor), and one PNET cell line (TC32) were found to strongly express the SgII gene, as shown by the presence of specific mRNA. This result supports the hypothesis that some Ewing's sarcomas represent a most primitive form of neuroectodermal tumor; in addition, it indicates a diagnostic role of SgII in cases of Ewing's sarcomas and PNETs.