RGS2 Expression Research Articles

Ischemia induces regulator of G protein signaling 2 (RGS2) protein upregulation and enhances apoptosis in astrocytes

Regulator of G protein signaling (RGS) family members, such as RGS2, interact with Galpha subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4-6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemia-induced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKCdelta, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKCdelta downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKCdelta mRNA and protein expressions. Ischemia-induced PKCdelta phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKCdelta knockdown, and this endogenous PKCdelta suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKCdelta and p38 MAPK pathways.

Cloning and characterization of rabbit Rgs4 promoter in gut smooth muscle

Regulator of G-protein signaling 4 (Rgs4) regulates the strength and duration of G-protein signaling, and plays an important role in cardiac development, smooth muscle contraction and psychiatric disorders. Rgs4 expression is regulated at both mRNA and protein levels. In order to examine the transcriptional mechanism of Rgs4 expression, we have cloned and characterized rabbit Rgs4 promoter. The coding sequence of rabbit Rgs4 was obtained by degenerative RT-PCR and used for Northern blot and 5′-RACE analysis. A single transcript was identified in rabbit colonic smooth muscle cells. The 5′-untranslated region (UTR) extended 120 bp nucleotides upstream of the Rgs4 start codon. A putative promoter sequence (1389 bp) showed a consensus TATA box and cis-acting binding sites for several potential transcriptional factors. Reporter gene assay identified strong promoter activity in various cell types. Further analysis by deletion mutagenesis suggested that the proximal region had a highest core promoter activity while the distal region is suppressive. IL-1β significantly increased the promoter activity. The in vitro and in vivo binding activities for NF-κB transcription factor were validated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay respectively. Mutation of NF-κB site reduced the promoter activity. These data suggest that the cloned rabbit Rgs4 promoter is functionally active and NF-κB binding site possesses enhancer activity in regulating Rgs4 transcription. Our studies provide an important basis for further understanding of Rgs4 regulation and function in different diseases.

Abstract 5785: A Novel Function of Regulator of G-protein Signaling 2: Stabilization of Signal Transducer and Activator of Transcription 3 Phosphorylation

Background: Regulator of G protein signaling proteins (RGS) are known to regulate G protein coupled receptor (GPCR) signaling at the plasma membrane through their GTPase-activating protein activity. We have previously identified RGS2 as a downstream target of unphosphorylated Signal Transducer and Activator of Transcription 3 (U-STAT3). U-STAT3 mediated Angiotensin II (AngII) type 1 receptor (AT1R), a GPCR, induced cardiac hypertrophy but the mechanism is unclear. Hypothesis: RGS2 is the mediator of U-STAT3 regulated Ang II/AT1R signaling. Methods and Results: In AT1R overexpressing HEK293 cells and HL-1 cells (a mouse cardiomyocyte cell line), Ang II stimulation induced RGS2 expression in parallel with U-STAT3. The localization, distribution and expression of RGS2 were changed upon Ang II stimulation. RGS2 was mainly localized in the nucleus, which was also confirmed in human vascular smooth muscle cells. Gene transfection of RGS2 dose-dependently increased phosphorylated STAT3 (P-STAT3), and maintained a high level of P-STAT3 up to 24 hrs. Co-immunoprecipitation assay identified a complex formed between RGS2 and P-STAT3, suggesting that RGS2 plays a role in maintaining P-STAT3 stability. Treatment with AT1R antagonist, losartan, decreased RGS2/P-STAT3 interaction, suggesting that this interaction is dependent on AT1R activation. On the other hand, in cells overexpressing AT1R and Y705F mutant STAT3, which cannot be phosphorylated on residue 705 (U-STAT3), chromatin immunoprecipitation assay demonstrated that Y705F mutant STAT3 binds to a consensus GAS motif located in the RGS2 promoter, providing direct evidence that RGS2 is a downstream target of U-STAT3. siRNA-mediated gene knockdown of STAT3 consistently caused decreased RGS2 expression in AT1R overexpressing cells. Conclusions: Chronic activation of AT1R induced U-STAT3 overexpression and subsequently induced overexpression of RGS2, which interacted with and stabilized P-STAT3. P-STAT3 further increases the transcription of stat3 gene, forming a positive feedback loop. We reported for the first time a novel function of RGS2, stabilization of P-STAT3, in mediating U-STAT3 regulated Ang II/AT1R signaling. Further studies are now underway utilizing engineered mice model. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Generation and characterization of a novel, cardiac‐specific, inducible RGS2 transgenic mouse model to modulate Gq‐mediated cardiac hypertrophy

Receptor signaling via heterotrimeric Gq proteins leads to hypertrophy, which is a risk factor for heart failure. Among Regulators of G‐protein Signaling (RGS), only RGS2 selectively inhibits Gq signaling and hypertrophy in cardiomyocytes in vitro. Our objective was to generate a mouse model that is suitable to test the hypothesis that RGS2 can modulate hypertrophy development and reversal in vivo. Using a doxycycline (Dox)‐responsive α myosin heavy chain promoter, we generated transgenic mice with cardiac‐specific, conditional RGS2 expression (TG) and established a Dox regimen that prevents RGS2 expression during development and ensures re‐expression by adulthood. Transgenic RGS2 was restricted to the myocardium, localized to both the particulate and cytosolic fraction, and dose‐dependently regulated by Dox in two independent lines. RGS2 expression did not alter heart weight, morphology and function under baseline conditions. Importantly, Gq‐mediated phospholipase Cβ activation was diminished in RGS2‐expressing ventricles. Our data indicate proper expression, regulation and function of transgenic RGS2 with no compensatory changes in other RGS proteins. Thus, this novel transgenic mouse model can now be used to determine the effect of RGS2 on Gq‐mediated pressure overload hypertrophy in vivo, using echocardiography and hemodynamic measurements.Supported by NIH grant HL80127.

Up‐regulation of RGS2 Mediate IL‐1beta‐Induced Decrease in Muscle Contraction in Response to NPR‐C activation in Tenia Coli

The proinflammatory cytokine, interleukine‐1β (IL‐1β), plays an important role in inflammation‐induced changes in smooth muscle contraction. IL‐1β inhibits acetylcholine release and muscle contraction in response to Gq‐coupled m3 receptor activation. Our previous studies have shown that the single‐transmembrane natriuretic peptide receptor, NPR‐C is coupled to Gi to stimulation of phosphoinositide (PI) hydrolysis via Gβγ i and muscle contraction.AIMTo characterize the effect of IL‐1β on contraction in response to NPR‐C activation.METHODSRabbit tenia coli were cultured in DMEM‐10 in the presence or absence of IL‐1β for 48 h. Muscle cells isolated from these strips were used for measurement of NPR‐C binding, PLC‐β3 and RGS2 expression, and cANP‐induced PI hydrolysis and muscle contraction.RESULTSPre‐incubation with IL‐1β had no effect on NPR‐C binding or PLC‐β3 expression, but significantly inhibited cANP‐induced PI hydrolysis and muscle contraction. IL‐1β caused a 3‐4 fold‐increase in RGS2, a GTPase‐activating protein that accelerates inactivation of Gαi‐GTP, implying that the decrease in PI hydrolysis and contraction were mediated by increased deactivation of Gαi and formation of Gαβ γtrimer.CONCLUSIONInhibition of cANP‐induced contraction by IL‐1β reflects inhibition of PI hydrolysis resulting from up‐regulation of RGS2 and rapid inactivation of Gαi and inhibition of Gβγ i‐mediated PI hydrolysis.

Transcriptional Activation of Regulator of G Protein Signaling 4 (RGS4) by Inverted CCAAT Box Element (ICE) Binding Factor NF‐YA Attenuates G Protein Signaling

RGS4 is a member of a family of proteins that play critical roles in attenuating GPCR signaling. Enriched in brain, RGS4 is linked to several pathologies, including Parkinson's disease. However, the mechanisms underlying regulation of RGS4 expression have not been studied. Here, we elucidated these mechanisms in PC6 cells, in which RGS4 is induced markedly at cellular confluence. Using gene promoter analyses, we demonstrated that an ICE in the human RGS4 promoter was required for transcriptional activation of RGS4. Phylogenetic analysis revealed that the ICE was one of four conserved transcription factor binding sites. Functionally, the ICE interacted with NF‐YA, an ICE binding transcription factor, in EMSA and biotin‐labeled DNA oligo pull‐down assays. Exogenous expression of NF‐YA or C/EBPβ activated the RGS4 promoter, while dominant negative NF‐YA and C/EBPβ significantly reduced RGS4 promoter activity. Furthermore, both cyclin D1 and phosphorylated Rb were decreased in confluent cells, indicating cell growth arrest. However, growth arrest induced by serum withdrawal alone did not alter RGS4 expression. Consistent with the fact that RGS4 is a GAP for Gαi and Gαq, LPA‐induced activation of MAPK was attenuated in confluent cells. We conclude that transcriptional activation of RGS4 by NF‐YA leads to an attenuation of G‐protein signaling. Supported by grants from the NIH (GM075033) and AHA (0750057Z).

IL-17 activates the classical NF-κB signaling pathway to upregulate chemokine signal inhibitor molecules RGS16 and RGS13 in autoimmune B cells of BXD2 mice (99.49)

Abstract We have previously reported that IL-17 upregulated the expression of rgs13 and rgs16 to attest the migration response of B cell to CXCL12. This specific effect of IL-17 on B cells is associated with the development of spontaneous germinal center in the spleens of autoimmune BXD2 mice. This study is to determine if NF-κB signaling is involved in IL-17 mediated upregulation of RGS proteins in B cells. Western blotting and confocal imaging analyses both showed that IL-17 stimulation of B cells from BXD2 mice resulted in rapid (5 min) activation of the classical NF-κB pathway which can be characterized by phosphorylation of p65 (P-p65) and translocation of P-p65 to the nuclei. Significantly higher level of activated P-p65 was found in BXD2 B cells than in B6 and BXD2-Il17r-/- B cells (>4.0-fold, p<0.01). Expression of the endogenous p50 subunit of NF-κB in BXD2 B cells was also higher than in B6 B cells (5.0-fold, p<0.01) but was surprisingly lower than in BXD2-Il17r-/- B cells (2.5-fold, p<0.01). The activated NF-κB pathway by IL-17 in B cells correlated with the upregulation of RGS16 and RGS13 in both protein and mRNA levels. Inhibition of P-p65 significantly down-regulated the expression of rgs genes (6.0-fold), and released the B cell migration arrest induced by IL-17 stimulation. These data suggest that IL-17 directly upregulates the expression of RGS through activating the classical NF-κB pathway in autoreactive BXD2 B cells.

A Potential Protective Role of RGS2 in Oxidative‐Stress Mediated Anxious Behavior in Rats

A single nucleotide polymorphism in Regulator of G‐Protein Signaling (RGS)‐2 gene in human and its knockout in mice have been linked to panic disorder and anxiety, respectively. Interestingly, oxidative stress is proposed to be involved in anxiety. The present study is the first to provide a link between RGS2, oxidative stress and anxiety. Our neuronal cell culture studies demonstrate that 1h H2O2 treatment increases while 4h H2O2 treatment decreases RGS2 and antioxidant proteins. RGS2 antisense treatment prevented the increase in antioxidant protein expression suggesting the involvement of RGS2 in this process. Oxidant producing pharmacological agent BSO treatment for 4 and 7 d in rats, increased the levels of oxidative stress markers, protein nitrotyrosine and MDA, in the brain regions implicated in the anxiety response. This was accompanied by a decrease in RGS2 and antioxidant proteins especially, glutathione reducatse 1 and Glyoxalase 1, the proteins previously implicated in the development of anxiety. Two day BSO treatment increased RGS2 and antioxidant protein expression. Furthermore, behavior tests revealed that 4 and 7 d BSO treated rats were more anxious than control or antioxidant, tempol treated rats while 2d treatment did not result in increased anxiety. Our studies suggest an involvement of oxidative stress in anxiety and a novel role of RGS2 in antioxidant homeostasis. [Funding: GEAR, UH].

Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate μ- and δ-opioid receptor signaling

In vitro studies have shown that the Regulator of G protein Signaling 4 (RGS4) interacts with the C-termini of μ- and δ-opioid receptors (μ-OR, δ-OR) (Georgoussi et al., 2006, Cell. Signal.18, 771–782). Herein we demonstrate that RGS4 associates with these receptors in living cells and forms selective complexes with Gi/Go proteins in a receptor dependent manner. This interaction occurs within the predicted fourth intracellular loop of μ, δ-ORs as part of a signaling complex consisting of the opioid receptor, activated Gα and RGS4. RGS4 is recruited to the plasma membrane upon opioid receptor stimulation. Expression of RGS4 in HEK293 cells attenuated agonist-mediated extracellular signal regulated kinase (ERK1,2) phosphorylation for both receptors and accelerated agonist-induced internalization of the δ-OR. RGS4 lacking its N-terminal domain failed to interact with both opioid receptors and to modulate opioid receptor signaling. Our findings demonstrate that RGS4 plays a key role in G protein coupling selectivity and signaling of the μ- and δ-ΟRs.

RGS17, an Overexpressed Gene in Human Lung and Prostate Cancer, Induces Tumor Cell Proliferation Through the Cyclic AMP-PKA-CREB Pathway

We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth.

Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells

Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P 1/S1P 3–5 agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was G i/o-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.

Mapping the Regulator of G Protein Signaling 4 (RGS4): Presynaptic and Postsynaptic Substrates for Neuroregulation in Prefrontal Cortex

Regulator of G protein signaling 4 (RGS4) regulates intracellular signaling via G proteins and is markedly reduced in the prefrontal cortex (PFC) of patients with schizophrenia. Characterizing the expression of RGS4 within individual neuronal compartments is thus key to understanding its actions on individual G protein-coupled receptors (GPCRs). Here we present an ultrastructural reference map of RGS4 protein in macaque PFC based on immunogold electron microscopic analysis. At the soma, all labeling was asynaptic and affiliated with subsurface cistern microdomains of pyramidal neurons. The nucleus displayed most of immunoreactivity. RGS4 levels were particularly high along proximal apical dendrites and markedly decreased with distance from the soma; clustered label was present at the bifurcation into second-order branches. In distal dendrites and in spines, the protein was found flanking or directly facing the postsynaptic density of symmetric and asymmetric synapses. Axons also expressed RGS4. In fact, the density and distribution of pre- and postsynaptic labeling was correlated with the axon ultrastructure and the type of established synapses. The data indicate that RGS4 is strategically positioned to regulate not only postsynaptic but also presynaptic signaling in response to synaptic and nonsynaptic GPCR activation, having broad yet highly selective influences on multiple aspects of PFC cellular physiology.

Regulators of G‐protein signalling are modulated by bacterial lipopeptides and lipopolysaccharide

Regulators of G-protein signalling accelerate the GTPase activity of G(alpha) subunits, driving G proteins in their inactive GDP-bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested. Real-time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway. This was confirmed using MyD88(-/-) and TRIF(-/-) bone marrow-derived macrophages. Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.

Runx2 Regulates G Protein-coupled Signaling Pathways to Control Growth of Osteoblast Progenitors

Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-beta/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G(1). Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2(-/-) osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals.

And the Winner Is … RGS4!

See related article, pages 527–535 RGS proteins play a major role in controlling G protein signaling. Cifelli et al1 show very high expression of RGS4 in sinoatrial (SA) node in the mouse. Of the 10 RGS proteins expressed supraventricularly, genetic disruption of RGS4 alone produces a profound slowing of I K,Ach turnoff kinetics in SA node and enhanced carbachol-induced bradycardia in vivo. This is accompanied by a loss of rapid desensitization of the I K,Ach current, suggesting a selective action of RGS4 in controlling prolonged vagal responses. This has implications for clinical conditions dependent on cholinergic signals such as atrial fibrillation (AF), vasovagal syncope, and possibly torsades de point. These results make RGS4 an intriguing candidate gene in certain arrhythmias. Approximately 12 years ago, the G protein and G protein–coupled receptor fields were rocked by the discovery of the RGS (regulators of G protein signaling) proteins.2,3 Here was a key regulatory protein family that inhibited G protein signaling but was virtually unknown a year earlier. Very rapidly, the core molecular function of RGS proteins was defined as GTPase accelerator protein activity, which sped the deactivating GTP hydrolysis step,4 which, for G proteins, was notoriously slow. The crystal structure of a complex of Gαi1 and RGS4 “caught in the act” of GTP hydrolysis5 followed shortly thereafter. Cardiac atrial function figured prominently in early studies of RGS proteins. The negative chronotropic actions of acetylcholine via the M2 muscarinic receptor involved activation of the I K,Ach current, which is carried by the G protein–coupled inwardly rectifying K+ (GIRK) channel, composed of a heterotetramer of subunits Kir3.1/Kir3.4. GIRK channels are activated by Gβγ subunits released from Gi/o family proteins. Vagal control of heart rate occurs on a very rapid times scale, with slowing and recovery within single …

Characterization of the transcriptional regulation of the regulator of G protein signaling 2 (RGS2) gene during 3T3‐L1 preadipocyte differentiation

Adipocyte differentiation is a complex process involving several signaling pathways. Molecular mechanisms regulating the very early stage of adipocyte differentiation is not fully appreciated yet. Several inducible genes at the early stage of preadipocyte differentiation have been identified, including the regulator of G protein signaling 2 (RGS2), a member of the RGS protein superfamily. This study aimed to clarify the precise induction profile of RGS2 and to determine the essential transcription element(s) regulating RGS2 expression in differentiating 3T3-L1 preadipocytes. RGS2 mRNA expression was elevated immediately at 1 h after differentiation initiation and it remained high until the late stage of differentiation. The putative promoter sequence (approximately 3,000 bp) of the mouse RGS2 gene was isolated and the RGS2 promoter activity was significantly upregulated 3 h after inducing differentiation. The primary signaling pathway leading to RGS2 transcriptional activation appeared to be cAMP-dependent. Sequential deletion and site-directed mutagenesis strategies demonstrate that the RGS2 promoter sequence truncated down to 78 bp in size retained full inducibility by the differentiation stimuli. Mutation of a Sp1 site within the 78 bp region significantly blocked promoter activity. In addition, high expression of Sp1 transcription factor was noted prior to and paralleling the differentiation process. Taken together, our data suggest that RGS2 transcription is immediately induced via a cAMP-dependent pathway after initiation of 3T3-L1 differentiation and the RGS2 mRNA level remains consistently high throughout the differentiation progression. A Sp1 site within RGS2 promoter appeared to be a crucial response element to regulate RGS2 transcription.

PLC‐β1 knockout mice as a model of disrupted cortical development and plasticity: Behavioral endophenotypes and dysregulation of RGS4 gene expression

The complexity of the genetics underlying schizophrenia is highlighted by the multitude of molecular pathways that have been reported to be disrupted in the disorder including muscarinic, serotonergic, and glutamatergic signaling systems. It is of interest, therefore, that phospholipase C-beta1 (PLC-beta1) acts as a point of convergence for these pathways during cortical development and plasticity. These signaling pathways, furthermore, are susceptible to modulation by RGS4, one of the more promising candidate genes for schizophrenia. PLC-beta1 knockout mice were behaviorally assessed on tests including fear conditioning, elevated plus maze, and the Y maze. In situ hybridization was used to assess RGS4 expression. We found that PLC-beta1 knockout mice display abnormal anxiety profiles on some, but not all measures assessed, including decreased anxiety on the elevated plus maze. We also show memory impairment and a complete absence of acquisition of hippocampal-dependent fear conditioning. Furthermore, at a molecular level, we demonstrate dramatic changes in expression of RGS4 mRNA in selective regions of the PLC-beta1 knockout mouse brain, particularly the CA1 region of the hippocampus. These results validate the utility of the PLC-beta1 knockout mouse as a model of schizophrenia, including molecular and cellular evidence for disrupted cortical maturation and associated behavioral endophenotypes.

Age-related changes in the expression of schizophrenia susceptibility genes in the human prefrontal cortex

The molecular basis of complex neuropsychiatric disorders most likely involves many genes. In recent years, specific genetic variations influencing risk for schizophrenia and other neuropsychiatric disorders have been reported. We have used custom DNA microarrays and qPCR to investigate the expression of putative schizophrenia susceptibility genes and related genes of interest in the normal human brain. Expression of 31 genes was measured in Brodmann's area 10 (BA10) in the prefrontal cortex of 72 postmortem brain samples spanning half a century of human aging (18-67 years), each without history of neuropsychiatric illness, neurological disease, or drug abuse. Examination of expression across age allowed the identification of genes whose expression patterns correlate with age, as well as genes that share common expression patterns and that possibly participate in common cellular mechanisms related to the emergence of schizophrenia in early adult life. The expression of GRM3 and RGS4 decreased across the entire age range surveyed, while that of PRODH and DARPP-32 was shown to increase with age. NRG1, ERBB3, and NGFR show expression changes during the years of greatest risk for the development of schizophrenia. Expression of FEZ1, GAD1, and RGS4 showed especially high correlation with one another, in addition to the strongest mean levels of absolute correlation with all other genes studied here. All microarray data are available at NCBI's Gene Expression Omnibus: GEO Series accession number GSE11546 (http://www.ncbi.nlm.nih.gov/geo) [corrected]

Lack of receptor-selective effects of either RGS2, RGS3 or RGS4 on muscarinic M 3- and gonadotropin-releasing hormone receptor-mediated signalling through Gα q/11

Termination of signalling by G-protein-coupled receptors requires inactivation of the Gα-subunits of heterotrimeric G-proteins and the re-association of Gα- and Gβγ-subunits. Inactivation of Gα-subunits is achieved by the hydrolysis of bound GTP by an intrinsic GTPase activity, which is considerably enhanced by GTPase activating proteins. Regulators of G-protein signalling (RGS) proteins are a large family of GTPase activating proteins, many of which have structures indicating roles beyond GTPase activating protein activity and suggesting that the identity of the RGS protein recruited may also be critical to other aspects of signalling. There is some evidence of selective effects of RGS proteins against different G-protein-coupled receptors coupling to the same signalling pathways and growing evidence of physical interactions between RGS proteins and G-protein-coupled receptors. However, it is unclear as to how common such interactions are and the circumstances under which they are functionally relevant. Here we have examined potential selectivity of RGS2, 3 and 4 against signalling mediated by Gα q/11-coupled muscarinic M 3 receptors and gonadotropin-releasing hormone in an immortalised mouse pituitary cell line. Despite major structural differences between these two receptor types and agonist-dependent phosphorylation of the muscarinic M 3- but not gonadotropin-releasing hormone receptor, signalling by both receptors was similarly inhibited by expression of either RGS2 or RGS3, whereas RGS4 has little effect. Thus, at least in these circumstances, RGS protein-dependent inhibition of signalling is not influenced by the nature of the G-protein-coupled receptor through which the signalling is mediated.

S1683 Regulation of Constitutive and IL-1β-Induced RGS4 Expression in Smooth Muscle Cells Is Mediated Via MEKK1-JNK-Ap1 Pathway

S1683 Regulation of Constitutive and IL-1β-Induced RGS4 Expression in Smooth Muscle Cells Is Mediated Via MEKK1-JNK-Ap1 Pathway