Oxidative stress and heat shock stimulate RGS2 expression in 1321N1 astrocytoma cells

RGS2, a regulators of G-protein signaling family member, regulates G-protein signaling and is itself controlled in part by regulated expression. We tested if cell stress regulates RGS2 expression in human astrocytoma 1321N1 cells. Treatment with H2O2 increased RGS2 mRNA levels time- and concentration-dependently, with 200 microM H2O2 causing an approximately eightfold increase after 2 h. Peroxynitrite and heat shock also increased RGS2 mRNA levels. H2O2-induced RGS2 expression was negatively regulated by phosphoinositide-3-kinase and extracellular signal-regulated kinases. H2O2 also concentration-dependently increased RGS2 protein levels, and the RGS2 appeared to be predominantly in the nucleus. These results demonstrate that RGS2 expression is up-regulated by cell stress.     

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.     

Regulation of longevity by regulator of G-protein signaling protein, Loco

Regulator of G-protein signaling (RGS) proteins contribute to G-protein signaling pathways as activators or repressors with GTPase-activating protein (GAP) activity. To characterize whether regulation of RGS proteins influences longevity in several species, we measured stress responses and lifespan of RGS-overexpressing and RGS-lacking mutants. Reduced expression of Loco, a RGS protein of Drosophila melanogaster, resulted in a longer lifespan for both male and female flies, also exhibiting stronger resistance to three different stressors (starvation, oxidation, and heat) and higher manganese-containing superoxide dismutase (MnSOD) activity. In addition, this reduction in Loco expression increased fat content and diminished cAMP levels. In contrast, overexpression of both genomic and cDNA loco gene significantly shortened the lifespan with weaker stress resistance and lower fat content. Deletion analysis of the Loco demonstrated that its RGS domain is required for the regulation of longevity. Consistently, when expression of RGS14, mammalian homologue of Loco, was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression. The changes of yeast Rgs2 expression, which shares a conserved RGS domain with the fly Loco protein, also altered lifespan and stress resistance in Saccharomyces cerevisiae. Here, we provide the first evidence that RGS proteins with GAP activity affect both stress resistance and longevity in several species.     

YTHDF2 Suppresses Notch Signaling through Post-transcriptional Regulation on Notch1

YTH domain family 2 (YTHDF2) is an N6-methyladenosine (m⁶A) binding protein promoting mRNA degradation in various biological processes. Despite its essential roles, the role of YTHDF2 in determining cell fates has not been fully elucidated. Notch signaling plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. We investigated the effect of YTHDF2 on Notch signaling. Our results show that YTHDF2 inhibits Notch signaling by downregulating the Notch1, HES1, and HES5 mRNA levels. Analyzing YTHDF2 deletion mutants indicates that the YTH domain is critical in regulating the Notch signal by directly binding m⁶A of Notch1 mRNA. Recently, YTHDF2 nuclear translocation was reported under heat shock conditions, but its physiological function is unknown. In our study, the YTH domain is required for YTHDF2 nuclear translocation. In addition, under heat shock stress, the Notch signal was significantly restored due to the increased expression of the Notch1 targets. These results suggest that YTHDF2 in the cytoplasm may act as an intrinsic suppressor in Notch signaling by promoting Notch1 mRNA degradation under normal cellular conditions. Conversely, upon the extracellular stress such as heat shock, YTHDF2 nuclear translocation resulting in reduced Notch1 mRNA decay may contribute to the increasing of Notch intracellular domain (NICD) regulating the survival-related target genes.     

Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells

Regulators of G protein signaling (RGS proteins) interact with Galpha(q) and Galpha(i) and accelerate GTPase activity. These proteins have been characterized only within the past few years, so our understanding of their importance is still preliminary. We examined the effect of oxytocin on RGS2 mRNA expression to help determine the role of RGS proteins in oxytocin signaling in human myometrial cells in primary culture. Oxytocin increased RGS2 mRNA concentration maximally by 1 or 2 h in a dose-dependent and agonist-specific manner. RGS2 mRNA levels were also elevated by treatment with Ca(2+) ionophore, phorbol ester, or forskolin. Oxytocin's effects were completely inhibited by an intracellular Ca(2+) chelator and partially blocked by a protein kinase C inhibitor, indicating that intracellular Ca(2+) concentration is the primary signal for oxytocin elevation of RGS2 mRNA levels. Use of pharmacological inhibitors indicated that part of oxytocin-stimulated RGS2 mRNA expression is mediated by G(i)/tyrosine kinase activities. Although oxytocin does not stimulate increases in intracellular cAMP concentration, agents that elevate intracellular cAMP concentrations and cause myometrial relaxation may possibly cause heterologous desensitization to oxytocin via RGS2 expression. These results suggest that RGS2 may be important in regulating the myometrial response to oxytocin.     

Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures

G protein-coupled receptor (GPCR) signaling cascades may be key substrates for the antidepressant effects of chronic electroconvulsive seizures (ECS). To better understand changes in these signaling pathways, alterations in levels of mRNA's encoding regulators of G protein signaling (RGS) protein subtypes-2, -4, -7, -8 and -10 were evaluated in rat brain using northern blotting and in situ hybridization. In prefrontal cortex, RGS2 mRNA levels were increased several-fold 2 h following an acute ECS. Increases in RGS8 mRNA were of lesser magnitude (30%), and no changes were evident for the other RGS subtypes. At 24 h following a chronic ECS regimen, RGS4, -7, and -10 mRNA levels were reduced by 20-30%; only RGS10 was significantly reduced 24 h after acute ECS. Levels of RGS2 mRNA were unchanged 24 h following either acute or chronic ECS. In hippocampus, RGS2 mRNA levels were markedly increased 2 h following acute ECS. More modest increases were seen for RGS4 mRNA expression, whereas levels of the other RGS subtypes were unaltered. At 24 h following chronic ECS, RGS7, -8 and -10 mRNA levels were decreased in the granule cell layer, and RGS7 and -8 mRNA levels were decreased in the pyramidal cell layers. Only RGS8 and -10 mRNA levels were significantly reduced in hippocampus 24 h following an acute ECS. Paralleling neocortex, RGS2 mRNA content was unchanged in hippocampus 24 h following either acute or chronic ECS. In ventromedial hypothalamus, RGS4 mRNA content was increased 24 h following chronic ECS, whereas RGS7 mRNA levels were only increased 24 h following an acute ECS. The increased RGS4 mRNA levels in hypothalamus were significant by 2 h following an acute ECS. These studies demonstrate subtype-, time-, and region-specific regulation of RGS proteins by ECS, adaptations that may contribute to the antidepressant effects of this treatment.     

Splicing Factor Transformer-2β (Tra2β) Regulates the Expression of Regulator of G Protein Signaling 4 (RGS4) Gene and Is Induced by Morphine

Regulator of G protein signaling 4 (RGS4) is a critical modulator of G protein-coupled receptor (GPCR)-mediated signaling and plays important roles in many neural process and diseases. Particularly, drug-induced alteration in RGS4 protein levels is associated with acute and chronic effects of drugs of abuse. However, the precise mechanism underlying the regulation of RGS4 expression is largely unknown. Here, we demonstrated that the expression of RGS4 gene was subject to regulation by alternative splicing of the exon 6. Transformer-2β (Tra2β), an important splicing factor, bound to RGS4 mRNA and increased the relative level of RGS4-1 mRNA isoform by enhancing the inclusion of exon 6. Meanwhile, Tra2β increased the expression of full-length RGS4 protein. In rat brain, Tra2β was co-localized with RGS4 in multiple opioid action-related brain regions. In addition, the acute and chronic morphine treatment induced alteration in the expression level of Tra2β in rat locus coerulus (LC) in parallel to that of RGS4 proteins. It suggests that induction of this splicing factor may contribute to the change of RGS4 level elicited by morphine. Taken together, the results provide the evidence demonstrating the function of Tra2β as a new mediator in opioid-induced signaling pathway via regulating RGS4 expression.     

Role of regulator of G-protein signaling 2 (RGS2) in periodontal ligament cells under mechanical stress

Mechanical stress is thought to regulate the expression of genes in the periodontal ligament (PDL) cells. Using a microarray approach, we recently identified a regulator of G-protein signaling 2 (RGS2) as an up-regulated gene in the PDL cells under compressive force. The RGS protein family is known to turn off G-protein signaling. G-protein signaling involves the production of cAMP, which is thought to be one of the biological mediators in response to mechanical stress. Here, we investigated the role of RGS2 in the PDL cells under mechanical stress. PDL cells derived from the ligament tissues of human premolar teeth were cultured in collagen gels and subjected to static compressive force. Compressive force application time-dependently enhanced RGS2 expression and intracellular cAMP levels. To examine the interrelationship between RGS2 and cAMP, the PDL cells were treated with 2',5'-dideoxyadenosine (DDA), an inhibitor of adenyl cyclase, or antisense S-oligonucleotide (S-ODN) to RGS2 under compressive force. DDA dose-dependently inhibited RGS2 stimulated by compressive force. Blockage of RGS2 by antisense S-ODN elevated the cAMP levels compared with controls. These results indicate that cAMP stimulates RGS2 expression, which in turn leads to a decrease in the cAMP production by inactivating the G-protein signaling in the mechanically stressed PDL cells.     

Muscarinic receptor stimulation increases regulators of G-protein signaling 2 mRNA levels through a protein kinase C-dependent mechanism.

RGS2, a member of the Regulators of G-protein Signaling family, modulates the activity of G-proteins coupled to the phosphoinositide signal transduction system, but little is known about what regulates RGS2. In human neuroblastoma SH-SY5Y cells stimulation of muscarinic receptors by carbachol activates phosphoinositide signaling and also caused a rapid, large, and long lasting increase in RGS2 mRNA levels. Direct activation of protein kinase C also rapidly increased RGS2 mRNA levels. Inhibition of protein kinase C with Ro31-8220, GF109203x, or Go6976 or down-regulation of protein kinase C inhibited increases in RGS2 mRNA levels induced by carbachol or by the activation of protein kinase C. Blockade of calcium signaling did not alter carbachol-induced increases in RGS2 mRNA levels. Neither activation of epidermal growth factor receptors nor stimulation of cyclic AMP production with forskolin increased RGS2 mRNA levels. Pretreatment with actinomycin D blocked increases in RGS2 mRNA levels but caused a surprisingly small, although statistically significant, partial blockade of protein kinase C-mediated feedback inhibition of carbachol-induced phosphoinositide hydrolysis. Thus, RGS2 mRNA levels are increased by activation of muscarinic receptors coupled to the phosphoinositide signal transduction system through a protein kinase C-dependent mechanism. This action may contribute to negative feedback control of this signaling cascade, but because the small contribution to negative feedback contrasts with the large and prolonged elevations in RGS2 mRNA levels, we speculate that its primary role may be in modulating other signaling components.     

RGS8 expression in developing cerebellar Purkinje cells

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 belongs to B/R4 subfamily of RGS proteins and is specifically expressed in Purkinje cells of adult cerebellum. Here, to examine the expression of RGS8 mRNA in developing cerebellum, we performed in situ hybridization. Apparent signals for expression of RGS8 mRNA were first detected on day 9 after birth, then RGS8 mRNA expression in Purkinje cells increased up to day 21, and its levels decreased to some extent in adult Purkinje cells. We also studied the expression of RGS7, which is expressed in Golgi cells in the granule cell layer of adult cerebellum. The expression of RGS7 mRNA was recognized in 7 day neonatal cerebellum. When examined with anti-RGS8 antibody, the RGS8 protein was already excluded from nucleus on day 9, and was distributed in cell body and dendrites in differentiating Purkinje cells of 14 day neonates.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.