Regulation of secretogranin II mRNA in rat neuronal cultures

doi:10.1016/0169-328X(95)00161-K

Molecular Brain Research

Volume 33, Issue 2, November 1995, Pages 326-332

https://doi.org/10.1016/0169-328X(95)00161-K Get rights and content

Abstract

The regulation of SgII mRNA expression was investigated in primary cultures of neurons prepared from the hypothalamus and brainstem of 1-day-old rats. The administration of forskolin (FSK) resulted in a time- and dose-dependent increase in SgII mRNA expression, a 9-fold effect within 6 h being achieved with 10 μM FSK, which maximally increased cellular cAMP levels. SgII mRNA levels remained elevated for 24 h. Activation of protein kinase C with 100 nM phorbol 12-myristate 13-acetate (PMA) also increased SgII mRNA expression, although induction with PMA was slower and more moderate (3.8-fold above control after 24 h). Neither 10 μM 1,9-dideoxyforskolin nor 100 nM 4α-phorbol 12,13-didecanoate, inactive analogues of FSK and PMA respectively, had an effect on SgII mRNA. Depolarization of neuronal cultures with 50 mM KCl had a small and variable effect on SgII mRNA levels (1.8-fold above control) in neuronal cultures and did not influence induction with FSK. To investigate whether neuron-like regulation of SgII mRNA expression could be reproduced in PC12 cells, PC12 cells were treated with 100 nM nerve growth factor (NGF) for 7 days prior to challenge with FSK or PMA. Whereas NGF alone modestly increased SgII mRNA expression in PC12 cells (1.8-fold above control), it did not uncover a stimulatory effect of FSK or PMA. These studies indicate that SgII mRNA expression is enhanced by an increase in cellular cAMP and activation of protein kinase C in primary cultures of neurons and emphasize that SgII mRNA is regulated in a cell-specific manner.

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Early data demonstrated that the up-regulation of Scg2 is a characteristic of phenotypic differentiation after neurotrophic growth factor treatment. For example, neuronal differentiation induced by nerve growth factor in PC12 cells (Laslop and Tschernitz, 1992; Scammell et al., 1995) or human neuroblastoma cells treated with phorbol esters (Weiler et al., 1990) is accompanied by an increase in biosynthesis of Scg2. Furthermore, other neurotrophic factors such as basic fibroblast growth factor and epidermal growth factor can increase Scg2 expression in human neuroblastoma cells (Weiss et al., 2001).
Secretogranin-2 (SCG2) is a large precursor protein that is processed into several potentially bioactive peptides, with the 30–43 amino acid central domain called secretoneurin (SN) being clearly evolutionary conserved in vertebrates. Secretoneurin exerts a diverse array of biological functions including regulating nervous, endocrine, and immune systems in part due to its wide tissue distribution. Expressed in some neuroendocrine neurons and pituitary cells, SN is a stimulator of the synthesis and release of luteinizing hormone from both goldfish pituitary cells and the mouse LβT2 cell line. Neuroendocrine, paracrine and autocrine signaling pathways for the stimulation of luteinizing hormone release indicate hormone-like activities to regulate reproduction. Mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. A single injection of the SNa peptide enhanced reproductive outcomes in scg2a/scg2b double mutant zebrafish. Evidence in goldfish suggests a new role for SN to stimulate food intake by actions on other feeding-related neuropeptides. Expression and regulation of the Scg2a precursor mRNA in goldfish gut also supports a role in feeding. In rodent models, SN has trophic-like properties promoting both neuroprotection and neuronal plasticity and has chemoattractant properties that regulate neuroinflammation. Data obtained from several cellular models suggest that SN binds to and activates a G-protein coupled receptor (GPCR), but a bona fide SN receptor protein needs to be identified. Other signaling pathways for SN have been reported which provides alternatives to the GPCR hypothesis. These include AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase II in cardiomyocytes, phosphatidylinositol 3-kinase (PI3K) and Akt/Protein Kinase B (AKT, and MAPK in endothelial cells and Janus kinase 2/signal transducer and activator of transcription protein (JAK2-STAT) signaling in neurons. Some studies in cardiac cells provide evidence for cellular internalization of SN by an unknown mechanism. Many of the biological functions of SN remain to be fully characterized, which could lead to new and exciting applications.
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Evolutionarily conserved sequences are bounded by conserved cleavage sites and presumably generate bioactive peptides that have fundamental functions, while most other sequences show much less distinct homology. Changes in granin expression in numerous conditions and pathological states point to their functional importance in health and disease (Schäfer et al., 1994; Scammel et al., 1995; Shen and Gundlach, 1998; Kato et al., 2000; Wiedermann, 2000; Taupenot et al., 2003; Mahapatra et al., 2005). Granin gene knockout and knockdown mice have been developed only for CgA (Huh et al., 2003; Mahapatra et al., 2005; Hendy et al., 2006; Kim et al., 2006; Montesinos et al., 2008), and have demonstrated a major role for this granin in secretory granule biogenesis, neuropeptide sorting and catecholamine storage; constitutive CgA knockouts show compensatory overexpression of CgB and SgII in adrenal medullary cells, which suggest counter-regulatory mechanisms of granin expression.
Morphologically distinct neuron classes can be subdivided in sublineages by differential chemical phenotypes that correlate with functional diversity. Here we show by immunocytochemistry that chromogranin A (CgA) chromogranin B (CgB) and secretogranin II (SgII), the principal granins situated in neuronal secretory granules and large dense-core vesicles, are widely but differentially expressed in cells of the mouse cerebellum and terminals of cerebellar afferents. While CgA and CgB were nearly panneuronal, SgII was more restricted in distribution. The cells most intensely immunoreactive for SgII were a class of small, excitatory interneurons enriched in the granular layer of the vestibulocerebellum, the unipolar brush cells (UBCs), although larger neurons likely to be a subset of the Golgi–Lugaro–globular cell population were also distinctly immunopositive; by contrast, Purkinje cells and granule cells were, at best, faintly stained and, stellate, basket cells were unstained. SgII was also present in subsets of mossy fibers, climbing fibers and varicose fibers. Neurons in the cerebellar nuclei and inferior olive were distinctly positive for the three granins. Double-labeling with subset-specific cell class markers indicated that, while both CgA and CgB were present in most UBCs, SgII immunoreactivity was present in the calretinin (CR)-expressing subset, but lacked in metabotropic glutamate receptor 1alpha (mGluR1α)-expressing UBCs. Thus, we have identified an additional cell class marker, SgII, which serves to study subtype properties in the UBC population. The abundance of SgII in only one of the two known subsets of UBCs is remarkable, as its expression in other neurons of the cortex was moderate or altogether lacking. The data suggest that the CR-positive UBCs represent a unique neuroendocrine component of the mammalian cerebellar cortex, presumably endowed with transynaptically regulated autocrine or paracrine action/s. Because of the well-known organization of the cerebellar system, several of its neuron classes may represent valuable cellular models to analyze granin functions in situ, in acute slices and in dissociated cell and organotypic slice cultures.
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In support of previous data from this laboratory, acute amphetamine increased the expression of PPD, PPT, PPE, and SGII gene expression in the striatum [13,36,38]. The effects of amphetamine on the expression of these neuropeptides is triggered by D1 receptor stimulation of the cAMP/PKA cascade [8,33]. In addition, these events are modulated by numerous other neurotransmitter systems in the striatum.
The purpose of this study was to investigate whether GABA_B receptor activation blocks acute amphetamine-induced behavioral activity, dopamine release, and neuropeptide mRNA expression in the striatum. Systemic administration of R-(+)-baclofen (1.25 mg/kg, i.p.) did not alter total distance traveled or vertical rearing induced by amphetamine (2.5 mg/kg, i.p.). At 2.5 mg/kg, baclofen did not alter spontaneous motor activity or total distance traveled, but completely blocked vertical rearing induced by amphetamine. At 5.0 mg/kg, baclofen completely blocked both total distance traveled and vertical rearing induced by amphetamine. Quantitative in situ hybridization histochemistry revealed that baclofen (2.5 mg/kg, i.p.) decreased the ability of amphetamine to increase preprodynorphin (PPD), preprotachykinin (PPT), preproenkephalin (PPE), and secretogranin II (SGII) mRNA levels in the striatum without altering the basal levels of these signals. Baclofen also blocked the amphetamine-induced rise in SGII mRNA in the core and shell of the nucleus accumbens and cingulate cortex. In a separate experiment, systemic baclofen (2.5 mg/kg) decreased the amphetamine-induced increase in dialysate dopamine levels in the striatum. These results suggest that reduced striatal dopamine release contributes to the ability of GABA_B receptor activation to decrease acute amphetamine-induced behavioral activity and striatal neuropeptide gene expression.
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To investigate the events involved in regulation of the secretogranin II (SgII) gene, luciferase reporter constructs were transfected into gonadotrope-derived, αT3-1 cells. DNA between −91 and −60 relative to the transcription start site was found to be required for GnRH induced SgII reporter gene activation. This region contains a consensus cAMP response element (CRE) and disruption of this CRE reduced GnRH responsiveness of the SgII promoter. CREB was shown to bind to the SgII CRE and transfection studies with a dominant-negative CREB mutant provided evidence that CREB is required for GnRH responsiveness of the SgII promoter. An expression vector for an inhibitor of the cAMP-dependent protein kinase was found to reduce the ability of cAMP or GnRH to activate the SgII-luciferase reporter gene. These studies offer evidence that GnRH-induced activation of the SgII promoter in the αT3-1 cell line requires cAMP-dependent protein kinase activity and a functional CRE within the 5′-flanking region of the gene.
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Research reportRegulation of secretogranin II mRNA in rat neuronal cultures

Abstract

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Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

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p1B15: a cDNA clone of the rat mRNA encoding cyclophilin

DNA

Secretogranin II is synthesized and secreted in astrocyte cultures

J. Neurochem.

Research report
Regulation of secretogranin II mRNA in rat neuronal cultures