RGS4 Protein Research Articles

The antiepileptogenic effect of low-frequency stimulation on perforant path kindling involves changes in regulators of G-protein signaling in rat

G-protein coupled receptors may have a role in mediating the antiepileptogenic effect of low-frequency stimulation (LFS) on kindling acquisition. This effect is accompanied by changes at the intracellular level of cAMP. In the present study, the effect of rolipram as a phosphodiesterase inhibitor on the antiepileptogenic effect of LFS was investigated. Meanwhile, the expression of αs- and αi-subunit of G proteins and regulators of G-protein signaling (RGS) proteins following LFS application was measured. Male Wistar rats were kindled by perforant path stimulation in a semi-rapid kindling manner (12 stimulations per day) during a period of 6days. Application of LFS (0.1ms pulse duration at 1Hz, 200 pulses, 50–150μA, 5min after termination of daily kindling stimulations) to the perforant path retarded the kindling development and prevented the kindling-induced potentiation and kindling-induced changes in paired pulse indices in the dentate gyrus. Intra-cerebroventricular microinjection of rolipram (0.25μM) partially prevented these LFS effects. Twenty-four hours after the last kindling stimulation, the dentate gyrus was removed and changes in protein expression were measured by Western blotting. There was no significant difference in the expression of α-subunit of Gs and Gi/o proteins in different experimental groups. However, application of LFS during the kindling procedure decreased the expression RGS4 and RGS10 proteins (that reduce the activity of Gi/o) and prevented the kindling-induced decrease of RGS2 protein (which reduces the Gs activity). Therefore, it can be postulated that the Gi/o protein signaling pathways may be involved in antiepileptogenetic effect of LFS, and this is why decreasing the cAMP metabolism by rolipram attenuates this effect of LFS.

Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity.

RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.

An RGS2 3'UTR polymorphism is associated with preeclampsia in overweight women.

BackgroundPreeclampsia is a common and heterogeneous vascular syndrome of pregnancy. Its genetic risk profile is yet unknown and may vary between individuals and populations. The rs4606 3′ UTR polymorphism of the Regulator of G-protein signaling 2 gene (RGS2) in the mother has been implicated in preeclampsia as well as in the development of chronic hypertension after preeclampsia. The RGS2 protein acts as an inhibitor of physiological vasoconstrictive pathways, and a low RGS2 level is associated with hypertension and obesity, two conditions that predispose to preeclampsia. We genotyped the rs4606 polymorphism in 1339 preeclamptic patients and in 697 controls from the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort to study the association of the variant with preeclampsia.ResultsNo association between rs4606 and preeclampsia was detected in the analysis including all women. However, the polymorphism was associated with preeclampsia in a subgroup of overweight women (body mass index ≥ 25 kg/m2, and < 30 kg/m2) (dominant model; odds ratio, 1.64; 95 % confidence interval, 1.10–2.42).ConclusionsOur results suggest that RGS2 might be involved in the pathogenesis of preeclampsia particularly in overweight women and contribute to their increased risk for hypertension and other types of cardiovascular disease later in life.

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [35S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury.

Regulator of G protein signaling (RGS) proteins have emerged as novel drug targets since their discovery almost two decades ago. RGS2 has received particular interest in cardiovascular research due to its role in regulating Gqsignaling in the heart and vascular smooth muscle. RGS2(-/-)mice are hypertensive, prone to heart failure, and display accelerated kidney fibrosis. RGS2 is rapidly degraded through the proteasome, and human mutations leading to accelerated RGS2 protein degradation correlate with hypertension. Hence, stabilizing RGS2 protein expression could be a novel route in treating cardiovascular disease. We previously identified cardiotonic steroids, including digoxin, as selective stabilizers of RGS2 protein in vitro. In the current study we investigated the functional effects of digoxin-mediated RGS2 protein stabilization in vivo. Using freshly isolated myocytes from wild-type and RGS2(-/-)mice treated with vehicle or low-dose digoxin (2µg/kg/day for 7 days) we demonstrated that agonist-induced cAMP levels and cardiomyocyte contractility was inhibited by digoxin in wild-type but not in RGS2(-/-)mice. This inhibition was accompanied by an increase in RGS2 protein levels in cardiomyocytes as well as in whole heart tissue. Furthermore, digoxin had protective effects in a model of cardiac injury in wild-type mice and this protection was lost in RGS2(-/-)mice. Digoxin is the oldest known therapy for heart failure; however, beyond its activity at the Na(+)/K(+)-ATPase, the exact mechanism of action is not known. The current study adds a novel mechanism, whereby through stabilizing RGS2 protein levels digoxin could exert its protective effects in the failing heart.

Regulator of G-protein Signaling (RGS)1 and RGS10 Proteins as Potential Drug Targets for Neuroinflammatory and Neurodegenerative Diseases.

Regulator of G-protein signaling (RGS) proteins were originally identified as negative regulators of G-protein-coupled receptor (GPCR) signaling via their GTPase-accelerating protein (GAP) activity. All RGS proteins contain evolutionarily conserved RGS domain; however, they differ in their size and regulatory domains. RGS1 and RGS10 are smaller than other RGS proteins, and their functions involve various inflammatory responses including autoimmune responses in both the periphery and the central nervous system (CNS). Neuroinflammation is the chronic inflammatory response in the CNS. Acute inflammatory response in the CNS is believed to be beneficial by involving the neuroprotective actions of immune cells in the brain, particularly microglia, to limit tissue damage and to aid in neuronal repair. However, chronically elevated levels of cytokines serve to maintain activation of abundant numbers of immune cells potentiating prolonged inflammatory responses and creating an environment of oxidative stress, which further hastens oxidative damage of neurons. In this review, we describe the implications and features of RGS proteins (specifically RGS1 and RGS10) in neuroinflammation and neurodegenerative diseases. We will discuss the experimental and epidemiological evidence on the benefits of anti-inflammatory interventions by targeting RGS1 and/or RGS10 protein function or expression in order to delay or attenuate the progression of neurodegeneration, particularly in multiple sclerosis (MS) and Parkinson's disease (PD).

Regulator of G-protein signaling 4: A novel tumor suppressor with prognostic significance in non-small cell lung cancer

Regulator of G-protein signaling (RGS) family members are regulatory molecules which act as GTPase activating proteins (GAPs) for G alpha subunits of heterotrimeric G proteins. Emerging data indicated that RGS members were involved with tumorigenesis and metastasis. In the current study, we identified RGS4 as a novel tumor suppressor with prognostic significance in non-small cell lung cancer (NSCLC). To be specific, we found that RGS4 expression was higher in normal lung tissues than NSCLC specimens (P = 0.003). Further studies demonstrated that RGS4 was generally down-regulated in NSCLC specimens compared with the matched normal lung tissues, both at mRNA and protein levels. In addition, correlational analysis indicated that RGS4 expression levels negatively correlated with lymph node metastasis (P = 0.009) and TNM stage (P = 0.008). Survival analysis demonstrated that patients with lower RGS4 protein expression exhibited a much worse 5-year overall survival and 5-year disease-free survival than those with high expression. More importantly, we proved that over-expression of RGS4 in NSCLC cells decreased invasion and migration due to inhibition of MMP2/9 and reversal of EMT while down-regulation of RGS4 in normal lung cell lines promoted invasion and migration. At last, nude mice metastatic model proved that over-expression of RGS4 suppressed tumor metastasis in vivo. All of these results confirmed the critical role of RGS4 in NSCLC progression.

Reciprocal Alterations in Regulator of G Protein Signaling 4 and microRNA16 in Schizophrenia.

N-methyl-d-aspartate receptor (NMDAR) hypofunction in the dorsolateral prefrontal cortex (DLPFC) has been implicated in the pathology of schizophrenia. NMDAR activity is negatively regulated by some G protein-coupled receptors (GPCRs). Signaling through these GPCRs is reduced by Regulator of G protein Signaling 4 (RGS4). Thus, lower levels of RGS4 would enhance GPCR-mediated reductions in NMDAR activity and could contribute to NMDAR hypofunction in schizophrenia. In this study, we quantified RGS4 mRNA and protein levels at several levels of resolution in the DLPFC from subjects with schizophrenia and matched healthy comparison subjects. To investigate molecular mechanisms that could contribute to altered RGS4 levels, we quantified levels of small noncoding RNAs, known as microRNAs (miRs), which regulate RGS4 mRNA integrity after transcription. RGS4 mRNA and protein levels were significantly lower in schizophrenia subjects and were positively correlated across all subjects. The RGS4 mRNA deficit was present in pyramidal neurons of DLPFC layers 3 and 5 of the schizophrenia subjects. In contrast, levels of miR16 were significantly higher in the DLPFC of schizophrenia subjects, and higher miR16 levels predicted lower RGS4 mRNA levels. These findings provide convergent evidence of lower RGS4 mRNA and protein levels in schizophrenia that may result from increased expression of miR16. Given the role of RGS4 in regulating GPCRs, and consequently the strength of NMDAR signaling, these findings could contribute to the molecular substrate for NMDAR hypofunction in DLPFC pyramidal cells in schizophrenia.

Abstract P194: A Novel Signalosome in the Alpha1a-Adrenergic Receptor and its Genetic Variant Signaling Pathway

Objectives : Human α 1 adrenergic receptors (AR), members of G protein-coupled receptor superfamily (GPCR) regulate blood pressure via smooth muscle cell (SMC) proliferation and vasoconstriction. We showed that α 1a AR-G247R (247R) genetic variant, identified in a hypertensive patient, constitutively couples to β-arrestin1/MMP/EGFR transactivation pathway leading to hyperproliferation in fibroblasts, cardiomyoblasts and SMCs. A scaffolding protein spinophilin (SPL) serves as a docking site for many regulatory proteins including RGS2 (negative R egulators of G -protein S ignaling). SPL also binds 3 rd intracellular loop of some GPCRs. Hypothesis: differential interaction of 247R or α 1a AR-WT (WT) with SPL/RGS2/β-arrestin signalosome mediates unique signaling of 247R and hyperproliferation in cardiovascular cells. Methods: Receptor-protein interactions were determined by co-immunoprecipitation from HEK293 cells expressing HA-α 1 ARs and full length Myc-SPL, its fragments or Flag-RGS2. Protein levels were analyzed by Western. SPL knockdown or RGS2 overexpression was performed using SPL-specific or scrambled siRNA or Flag-RGS2. Results: Our results reveal that in SMCs or cardiomyoblasts 247R but not WT upregulates endogenous SPL. SPL exhibits stronger (~2-fold) interaction with WT compared to 247R or α 1b , recruits RGS2 and inhibits receptor signaling. In contrast, weak SPL-247R interaction diminishes RGS2 inhibitory effect permitting hyperproliferation of 247R cells. SPL knockdown inhibits 247R-induced proliferation by allowing RGS2 directly bind to 247R as observed with RGS2 overexpression. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that in 247R cells SPL binds RGS2 preventing RGS2-247R interaction. Conclusions: We present SPL/RGS2/β-arrestin as a novel signalosome responsible for α 1a AR-247R genetic variant triggered hyperproliferation in different cardiovascular cells. We reveal that SPL regulates α 1a AR signaling by differentially binding WT or 247R receptors and recruiting RGS2 protein to the receptor. These novel findings unravel critical roles of SPL and RGS2 in α 1 AR signaling, as well as identify SPL as a potential novel target for treatment of α 1 AR-mediated cardiovascular disorders.

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells.

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

Cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer.

More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.

FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex.

The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.

RGS2 Protein Degradation is Mediated by a Novel Cullin 4B/F‐box 44 E3 Ligase Complex

Hypertension and heart failure are major health issues and there is a need to identify more effective treatments. Regulator of G protein Signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and regulates Gαq signaling. RGS2‐/‐ mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Over‐expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.RGS2 is rapidly degraded through the proteasome, but the specific enzymes involved are not known. Thus, the goal of the current study was to identify the molecular machinery responsible for RGS2 protein degradation. A siRNA screen to identify genes that regulate RGS2 protein levels identified components of a putative cullin‐RING E3 ligase (CRL). siRNA knock‐down of F‐box 44 (FBXO44) or cullin 4B (CUL4B), but not CUL4A or CUL1, led to a significant increase in RGS2 protein levels and stability. Overexpression of FBXO44 and CUL4B decreased RGS2 protein levels. FBXO44 associates with both RGS2, CUL4B and the adaptor protein DDB‐1 in cells resulting in a novel E3 ligase protein complex able to degrade RGS2. Studies are now underway to characterize the molecular mechanisms of the FBXO44‐RGS2 interaction.RGS proteins are difficult drug targets due to their mode of action being through protein‐protein interactions. Inhibiting a more druggable protein within the degradation pathway could be a way to increase RGS2 protein levels and function.

RGS4 Differentially Regulates Antidepressant and Locomotor Behaviors In Vivo

Evidence suggests the antidepressant actions of serotonin (5‐HT) are modulated by regulators of G protein signaling (RGS) proteins. Specifically, we have shown that upregulation of cortical RGS4 (mRNA and protein) occurs in response to genetic disruption of RGS protein activity at the GTP‐binding protein Gαi2 and following chronic administration of serotonin‐selective reuptake inhibitors (SSRIs). Genetic deletion of RGS4 (RGS4(‐/‐)) had no effect on basal antidepressant‐like behaviors or generalized locomotor activity. However, RGS4(‐/‐) mice exhibited decreased antidepressant‐like responses to the SSRI fluoxetine, whereas desipramine, a selective inhibitor of norepinephrine reuptake, was fully efficacious. Conversely, the regulation of locomotor activity by desipramine was augmented in RGS4(‐/‐) animals relative to vehicle‐treated controls. RGS4 expression had no effect on locomotor activity following fluoxetine administration. Overall, these data suggest altered expression of RGS4 differentially influence antidepressant‐like behaviors in animal models of mood and potentially the therapeutic effects of SSRIs and are consistent with studies showing RGS4 positively regulates the efficacy of serotonin‐mediated antidepressants.

RGS2 Repression Exacerbates Airway Hyperresponsiveness and Remodeling in Asthma

We recently reported that RGS2, a modulator of Gq protein-coupled receptors, is a key regulator of airway hyperresponsiveness (AHR), the hallmark of asthma. RGS2 protein levels in airway cells were...

PKC Activation Leads to Increased RGS2 Protein Levels

Regulator of G protein Signaling 2 (RGS2) is a member of a family of proteins that regulate G protein‐coupled receptor (GPCR) mediated signaling by accelerating GTP hydrolysis on active Gα subunits. RGS2 has been indicated to play an important role in regulating cardiovascular function. RGS2‐/‐ mice are hypertensive, are prone to heart failure and show increased responses to vasoconstrictors, such as Angiotensin II. Strategies to increase RGS2 protein levels could thus be a beneficial therapeutic strategy. More detailed information on how RGS2 protein levels are regulated would facilitate these drug discovery efforts.A previous high‐throughput cell‐based screen of natural product extracts identified Indolactam V (ILV), a known PKC activator, to selectively increase protein levels of RGS2. Utilizing the PathHunter ProLabel assay; a β‐galatosidase complementation assay that measures relative protein levels, we found that ILV, as well as phorbol 12‐myristate 13‐acetate (PMA), selectively increase RGS2 protein levels in a time and concentration dependent manner. This effect was blocked by PKC inhibitors indicating a mechanism mediated by PKC. Stimulation of a Gq coupled receptor also results in an increase in RGS2 protein, indicating that this could be a novel feedback mechanism for Gq signaling. This study is continuing to dissect the mechanism by which PKC activation leads to increased RGS2 protein levels. Overall, these studies will lead to increased understanding of how RGS2 protein levels are regulated.

Effects of Cigarette Smoke Extract and Nicotine on Regulator of G Protein Signaling-2 Expression in Human Airway Smooth Muscle and Bronchial Epithelial Cells

RationaleRecent studies have shown that reductions of Regulator of G protein signaling 2 (RGS2) are associated with and lead to airway hyperresponsiveness in asthma and cigarette-induced lung disease. Human airway smooth muscle cells (HASM) treated with low dose (0.1%) cigarette smoke extract (CSE) for 10 days had a reduction in both mRNA and protein RGS2 expression. We aimed to determine if acute exposure of CSE to HASM and human bronchial epithelial cells (HBEC), which express RGS2, also reduced RGS2 expression. Further, we tested whether acute nicotine exposure alters RGS2 expression in HASM.MethodsHASM and HBEC were exposed to regular or ultra-low nicotine CSE (5% or 1%) or nicotine (50 uM and 2 uM) for 24 hrs. RT-PCR and Western blot were used to evaluate RGS2 expression.ResultsAcute challenge of HASM or HBEC with either CSE or nicotine did not significantly affect RGS2 protein or mRNA expression.ConclusionsOur data suggest that acute exposure of HASM or HBEC to CSE, at either high or low doses does not significantly alter RGS2 expression, in contrast with chronic low dose exposure in HASM. Since RGS2 regulates airway hyperresponsiveness and controls many important epithelial and smooth muscle cell functions, further study on the effects of cigarette smoke and/or nicotine on airway RGS2 expression and function are warranted. RationaleRecent studies have shown that reductions of Regulator of G protein signaling 2 (RGS2) are associated with and lead to airway hyperresponsiveness in asthma and cigarette-induced lung disease. Human airway smooth muscle cells (HASM) treated with low dose (0.1%) cigarette smoke extract (CSE) for 10 days had a reduction in both mRNA and protein RGS2 expression. We aimed to determine if acute exposure of CSE to HASM and human bronchial epithelial cells (HBEC), which express RGS2, also reduced RGS2 expression. Further, we tested whether acute nicotine exposure alters RGS2 expression in HASM. Recent studies have shown that reductions of Regulator of G protein signaling 2 (RGS2) are associated with and lead to airway hyperresponsiveness in asthma and cigarette-induced lung disease. Human airway smooth muscle cells (HASM) treated with low dose (0.1%) cigarette smoke extract (CSE) for 10 days had a reduction in both mRNA and protein RGS2 expression. We aimed to determine if acute exposure of CSE to HASM and human bronchial epithelial cells (HBEC), which express RGS2, also reduced RGS2 expression. Further, we tested whether acute nicotine exposure alters RGS2 expression in HASM. MethodsHASM and HBEC were exposed to regular or ultra-low nicotine CSE (5% or 1%) or nicotine (50 uM and 2 uM) for 24 hrs. RT-PCR and Western blot were used to evaluate RGS2 expression. HASM and HBEC were exposed to regular or ultra-low nicotine CSE (5% or 1%) or nicotine (50 uM and 2 uM) for 24 hrs. RT-PCR and Western blot were used to evaluate RGS2 expression. ResultsAcute challenge of HASM or HBEC with either CSE or nicotine did not significantly affect RGS2 protein or mRNA expression. Acute challenge of HASM or HBEC with either CSE or nicotine did not significantly affect RGS2 protein or mRNA expression. ConclusionsOur data suggest that acute exposure of HASM or HBEC to CSE, at either high or low doses does not significantly alter RGS2 expression, in contrast with chronic low dose exposure in HASM. Since RGS2 regulates airway hyperresponsiveness and controls many important epithelial and smooth muscle cell functions, further study on the effects of cigarette smoke and/or nicotine on airway RGS2 expression and function are warranted. Our data suggest that acute exposure of HASM or HBEC to CSE, at either high or low doses does not significantly alter RGS2 expression, in contrast with chronic low dose exposure in HASM. Since RGS2 regulates airway hyperresponsiveness and controls many important epithelial and smooth muscle cell functions, further study on the effects of cigarette smoke and/or nicotine on airway RGS2 expression and function are warranted.

RGS2 Suppresses Breast Cancer Cell Growth via a MCPIP1-Dependent Pathway

Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.

Regulator of G-protein signaling 2 repression exacerbates airway hyper-responsiveness and remodeling in asthma.

G protein-coupled receptors (GPCRs) are important regulators of cell functions in asthma. We recently reported that regulator of G-protein signaling (RGS) 2, a selective modulator of Gq-coupled GPCRs, is a key regulator of airway hyper-responsiveness (AHR), the pathophysiologic hallmark of asthma. Because RGS2 protein levels in airway cells were significantly lower in patients with asthma compared with patients without asthma, we further investigated the potential pathological importance of RGS2 repression in asthma. The human RGS2 gene maps to chromosome 1q31. We first screened patients with asthma for RGS2 gene promoter single-nucleotide polymorphisms (SNPs) and found significant differences in the distribution of two RGS2 SNPs (A638G, rs2746071 and C395G, rs2746072) between patients with asthma and nonasthmatic subjects. These two SNPs are always associated with each other and have the same higher prevalence in patients with asthma (65%) as compared with nonasthmatic subjects (35%). Point mutations corresponding to these SNPs decrease RGS2 promoter activity by 44%. The importance of RGS2 down-regulation was then determined in an acute IL-13 mouse model of asthma. Intranasal administration of IL-13 in mice also decreased RGS2 expression in lungs by ∼50% and caused AHR. Although naive RGS2 knockout (KO) mice exhibit spontaneous AHR, acute IL-13 exposure further increased AHR in RGS2 KO mice. Loss of RGS2 also significantly enhanced IL-13-induced mouse airway remodeling, including peribronchial smooth muscle thickening and fibrosis, without effects on goblet cell hyperplasia or airway inflammation in mice. Thus, genetic variations and increased inflammatory cytokines can lead to RGS2 repression, which exacerbates AHR and airway remodeling in asthma.

RGS2 and RGS4 proteins: New modulators of the κ-opioid receptor signaling

Previous studies have shown that RGS4 associates with the C-termini of μ- and δ-opioid receptors in living cells and plays a key role in Gi/Go protein coupling selectivity and signalling of these receptors [12,20]. To deduce whether similar effects also occur for the κ-opioid receptor (κ-ΟR) and define the ability of members of the Regulators of G protein Signaling (RGS) of the B/R4 subfamily to interact with κ-ΟR subdomains we generated glutathione S-transferase fusion peptides encompassing the carboxyl-termini of κ-OR (κ-CT). Results from pull down experiments indicated that RGS2 and RGS4 directly interact within different domains of the κ-CT. Co-precipitation studies in living cells indicated that RGS2 and RGS4 associate with κ-ΟR constitutively and upon receptor activation and confer selectivity for coupling with a specific subset of G proteins. Expression of both members, RGS2 and/or RGS4, in 293F cells attenuated κ-agonist mediated-adenylyl cyclase inhibition and extracellular signal regulated kinase (ERK1,2) phosphorylation with a different amplitude in their modulatory effect in κ-ΟR signaling. Our findings demonstrate that RGS2 and RGS4 are new interacting partners that play key roles in G protein coupling to negatively regulate κ-ΟR signaling.